Oligodendrocyte progenitor cells are promoted in the neurogenic niches of the juvenile mouse brain during viral infection

Oligodendrocyte progenitor cells are promoted in the neurogenic niches of the juvenile mouse brain during viral infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Oligodendrocyte progenitor cells are promoted in the neurogenic niches of the juvenile mouse brain during viral infection Yashika Kamte, Chloe Potosnak, Natalie London, Vivek Singh, Kyah Thompson, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8563506/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Viruses can profoundly disturb myelination in the brain, leading to enduring neurological sequelae. The outcomes of neurotropic infections can be especially dire in younger children, in whom developmental myelination is underway. In some adult models of viral infection, demyelination is immune-mediated; however, it is unclear how an antiviral immune response impacts myelination in the young brain. Methods We investigated the outcomes of a neuron-restricted viral infection on developmental myelination in juvenile mice (10 days old), where only mature neurons are infectable by measles virus (MeV). The impact of neuronal MeV infection and the ensuing antiviral immune response on myelination was assessed during acute infection (9 days post-infection, dpi) and afterwards in surviving mice (90 dpi). We quantified myelin proteins and lipids in multiple brain regions, assessed oligodendrocyte development from the oligodendrocyte progenitor (OPC) stage to maturity, and measured neuronal markers associated with appropriate myelination and synapse formation. Results We found that (a) neuron-restricted viral infection was associated with short-term disruptions in myelin lipids and long-term disruptions in myelin proteins in multiple brain regions; (b) the OPC pool expanded in the neurogenic niches of the brain both during and after acute infection; (c) the expansion in the OPC pool originated from increased differentiation by neural stem cells (NSCs) rather than OPC proliferation; (d) oligodendrocyte maturation increased despite diminished myelination; and (e) expression of axonal and synaptic markers were compromised in surviving mice, although neuronal numbers were maintained in the hippocampus. Conclusions Our findings show a robust OPC response in the juvenile brain during viral infection, but this response ultimately fails to normalize myelination and neuronal markers in surviving mice. We speculate that these mechanisms partly underlie life-long neurological impairments in some survivors of childhood infections. oligodendrocyte progenitor cells oligodendrocytes neural stem cells measles virus antiviral immunity myelin demyelination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 INTRODUCTION Viral infections of the central nervous system (CNS) are important causes of demyelination, encephalitis, and enduring neurological sequelae in children 1 – 5 . Although substantial brain development occurs in utero , the brain undergoes extensive maturation in childhood, with myelination, synaptogenesis, and synaptic pruning occurring throughout adolescence and even into adulthood 6 – 11 . Viruses can disrupt these processes through direct infection of developing brain cells ( e.g. , neurons, oligodendrocytes) and/or through bystander damage by the ensuing immune response against the virus. Additionally, in young children (< 10 years), viral infections often precede the onset of acute disseminated encephalomyelitis (ADEM), an immune-mediated demyelinating disorder triggered by many different viruses ( e.g ., measles, coronaviruses, influenza) after peripheral infection 4 , 12 – 16 . Thus, the immune response plays a multifaceted role in the young brain, where it may simultaneously contain viral spread while disturbing ongoing myelination. In the CNS, the myelin sheath is produced by oligodendrocytes (OLs). Upon selection of a neuronal axon, the OL plasma membrane wraps around a segment of the axon in tight concentric layers to form the sheath. In comparison to other cellular membranes, the membranes in the myelin sheath are enriched in lipids and myelin-specific proteins (e.g., myelin basic protein (MBP) and proteolipid protein (PLP)) that help to compact and stabilize the concentric layers. Myelination insulates the axon to allow for the propagation of faster action potentials and provides metabolic support to the neuron, further supporting axonal structure and integrity 17 , 18 . In humans, myelination begins in the second trimester of gestation, peaks at 2–3 years of age, and continues into adolescence and adulthood, with varying kinetics depending upon the anatomical site 6 , 19 – 22 . Brain regions involved in sensory and motor function are myelinated early in childhood, while neocortical regions involved in higher-order cognition are myelinated into adulthood. Mice follow a similar temporal pattern, with a wave of immature OLs emerging by postnatal day 10 (P10), a peak of myelination between 2–4 weeks of age, and continuation into adulthood (2–8 months old) 6 , 23 – 25 . Overall, myelination is crucial to facilitate connections in the developing brain and for rapid and synchronized transfer of information 26 . OL numbers increase dramatically in childhood as myelination peaks, but OLs do not typically divide 27 – 30 . Rather, new OLs are produced by specialized stem cells known as oligodendrocyte precursor cells (OPCs), which commit to the OL lineage through a carefully orchestrated series of cues during developmental myelination. OPCs can originate from the proliferation of existing OPCs or the commitment of neural stem cells (NSCs) to the glial lineage. Indeed, OPCs are among the most highly proliferative cells in the CNS, with greater OPC proliferation in white matter regions than grey matter regions prior to differentiation. OPCs can respond to injury throughout life by proliferating and migrating to sites of damage, promoting glial scar formation, and supporting neuronal signaling, among other roles 31 – 33 . Despite their versatility, new OPCs are impeded in their differentiation into OLs in several neurological diseases in adults ( e.g. multiple sclerosis (MS), stroke, schizophrenia) 34 – 37 . These demyelinating diseases have provided insights into the pathological responses of adult OPCs. However, in the developing brain, the role of OPCs is poorly understood in neuroinflammatory diseases, including in the context of viral infections and the ensuing immune response. Measles virus (MeV) is associated with a rare but diverse array of CNS complications in children, including demyelinating illnesses. MeV has been shown to directly infect OLs and neurons in subacute sclerosing panencephalitis (SSPE), which is a lethal, persistent infection by MeV 38 , 39 . In patients who succumbed to MeV encephalomyelitis, multiple myelin markers (e.g., MBP and myelin associated glycoprotein (MAG)) were reduced although MeV antigen was undetectable in the brain, suggesting an immune-mediated attack on the myelin sheath 40 . These studies suggest that MeV can induce demyelinating disease through direct infection of brain cells or through neuroinflammation. However, the response of developing OPCs and OLs to viral inflammation in childhood, when myelination is at its peak, is unknown. We previously studied MeV neuropathogenesis at the juvenile age (postnatal day 10) using NSE-CD46 mice (CD46+). In CD46 + mice, the neuron-specific enolase (NSE) promoter drives the expression of the human CD46 gene, which encodes one of three MeV receptors 41 . In CD46 + mice, only mature neurons are infected by MeV, while other neural cells are spared from direct viral infection. In juvenile CD46 + mice, ~ 25% of mice developed neurological symptoms (e.g., altered gait, ocular symptoms, seizures) by two weeks post MeV-infection and succumbed, despite robust infiltration of immune cells and cytokine expression. The surviving juvenile mice (~ 75%) remained asymptomatic up to three months post-infection, but ultimately developed neurological symptoms by five months, indicating declining neurological function 42 . Surprisingly, in both symptomatic and asymptomatic juvenile mice, NSC numbers transiently declined during acute infection, which was attributed only in part to modest cell death and limited differentiation into immature neurons, suggesting that NSCs may be marshalled toward a glial fate during viral infection 42 . Given the progressive neurological decline in mice that survived MeV infection as juveniles, the aim of this study was to determine if developmental myelination was disrupted due to infection. Because MeV is restricted to neurons in the CD46 + model, OPCs/OLs are spared from direct infection, thus allowing us to examine the bystander responses of these cells to the microenvironment created by the antiviral immune response and virally-infected neurons 43 , 44 . Ultimately, we speculate that juvenile OPCs are mobilized in the inflammatory microenvironment in the brain, but cannot overcome disruptions in developmental myelination, leading to long-term neurological consequences even after resolution of the initial infection. METHODS Experimental animals and ethics statement CD46 + mice were maintained and treated according to the Institutional Animal Care and Use Committee of Duquesne University under approved protocols and the NIH Guide for the Care and Use of Laboratory Animals. The CD46 + mice were maintained on a 12h/12h light/dark cycle under controlled temperature conditions (22 ± 5°C) with free access to food and water. Paired mating cages were established to generate juvenile mice for all experiments. Both male and female mice were used for the experiments reported herein. Measles virus (MeV) infections Postnatal day 10 (P10) CD46 + juvenile mice were infected with measles virus (MeV)-Edmonston strain obtained from ATCC (American Type Culture Collection; Cat No: VR-24). The virus was passaged three times and plaque-assayed in Vero cells. The inoculum was diluted in sterile phosphate buffered saline (PBS) and administered intracerebrally with a 1cc syringe (BD cat. no. 309659) and 27 1/2−gauge needle (BD cat. no. 305109). On P10, mice were lightly anesthetized with isoflurane and injected with 20 µl of MeV (2x10 4 PFU) or with 20 µl of PBS as a negative control. The MeV-infected or PBS control mice were observed daily for signs of sickness and mortality, for up to 9- or 90-days post infection (dpi). Western blotting Western blotting On the indicated dpi, mice were deeply anesthetized with isoflurane inhalation until unresponsive to pain stimuli (toe pinch), followed by cervical dislocation. The hippocampus, subventricular zone (SVZ), cerebellum, and cortex of each mouse were collected and lysed in 1× Cell Lysis Buffer (Cell Signaling Technology; cat. no. 9803) with 1× Protease Inhibitor Cocktail (Sigma-Aldrich; cat. no. P8340) (20 µL lysis buffer per mg tissue). Brain lysates were stored at − 80°C until further processing. The protein concentration of each lysate was measured using the Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (ThermoFisher Scientific; cat. no. 23225) on a TECAN Infinite M1000 plate reader. For the hippocampus, cerebellum, and cortex samples, 20 µg of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For the SVZ, 10 µg of protein were used for 9 dpi samples, whereas 15 µg of protein were used for 90 dpi samples because the SVZ dissections yielded relatively small pieces of tissue. The gel was blotted onto Immobilon-FL PVDF Membrane (Millipore; cat. no. IPFL00010) for 35 mins (semi-dry transfer) and the membranes were blocked using either a 1:1 mixture of 1× PBS/Tween-20 solution (Sigma-Aldrich; cat. no. P3563) and Odyssey blocking buffer (LICOR Biosciences; cat. no. 927–40000) or undiluted Intercept (TBS) blocking buffer (LICOR Biosciences; cat. no. 927-60001) for 1 hour at room temperature. The membranes were treated with primary antibody diluted in the aforementioned blocking solution overnight at 4°C on a rocker. The membranes were washed thrice with PBS-Tween (10 mins/wash) and incubated for one hour at room temperature in secondary antibody solutions, including goat anti-rabbit 800 (LI-COR Biosciences; cat. no. 926–32211; 1:10,000), goat anti-rat 800 (LI-COR Biosciences; cat. no. 926-32219, 1:10,000), donkey anti-rabbit 680 (LI-COR Biosciences; cat. no. 926-68073, 1:10,000), and donkey anti-mouse 680 (LI-COR Biosciences; cat. no. 926–68072; 1:10,000). The membranes were then washed thrice (10 mins/wash) in PBS-Tween and imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences; model: Classic and M). Primary antibodies used were as follows: mouse anti-GAPDH (Millipore cat. no. MAB374; 1:10,000), rabbit anti-neurofascin186 (Cell Signaling Technology; cat. no. 15034s; 1:1000), rabbit anti-synapsin1/2 (Synaptic System; cat. no. 106002; 1:1000), rabbit anti-Caspr (Cell Signaling Technology cat. no. 97736S, 1:1000), rat anti-myelin basic protein (MBP; Millipore cat. no. MAB386, 1:500), rabbit anti-myelin proteolipid protein (PLP; Cell Signaling Technology cat. no. 28702, 1:1000), rabbit anti-myelin oligodendrocyte glycoprotein (MOG; Cell Signaling Technology cat. no. 96457T, 1:1000), and rabbit anti-myelin associated glycoprotein (MAG; Cell Signaling Technology cat. no. 9043T, 1:1000). FluoroMyelin Green staining Mice were lightly anesthetized with isoflurane either 9- or 90-days post infection, before intraperitoneal injection with 3.8% choral hydrate in PBS. This was followed by cardiac perfusion with ice-cold PBS followed by 4% paraformaldehyde/PBS (PFA/PBS; Affymetrix cat. no. 19943). Whole brains were collected, fixed with 4% PFA/PBS for 24 hours, and cryoprotected with 30% sucrose (Fisher Scientific cat. no. BP220–212) in PBS at 4°C. Brains were then sectioned on a sliding/freezing microtome in the sagittal plane at 40µm thickness as a 1-in-4 free floating series and stored at -20°C in cryoprotectant 45 . For staining brain slices with FluoroMyelin Green (ThermoFisher Scientific cat. no. F34651; 1:150), the manufacturer’s protocol was followed with minor modifications. Briefly, the tissue was washed in 10 mM PBS for 20 mins before staining them with FluoroMyelin green (1:150) for 20 mins at room temperature. The tissue sections were then washed three times for 10 mins in 10 mM PBS before mounting. ProLong Gold Antifade Mountant was used to mount FluoroMyelin-stained tissue (Thermo Fisher Scientific cat. no. P36930) on Superfrost plus slides (Fisher Scientific cat. no. 1255015). The areas of the hippocampus and corpus callosum were traced to quantify the FluoroMyelin green signal. The tissue was imaged on an Odyssey Infrared Imaging System (LI-COR Biosciences; Odyssey M model) at 5 µm resolution and on an epifluorescence microscope Olympus IX73 (B&B Microscopes). Tracings were performed using the Image Studio Lite Ver 5.2 (LI-COR Biosciences) to determine the total FluoroMyelin green signal, which was then expressed as a fraction of the trace area. PBS-injected controls and MeV infected animals were used (n = 4) at both 9 and 90 dpi. The hippocampus and corpus callosum from 10–20 slices per animal were traced after discarding torn slices. Flow cytometry for neural cells On the indicated dpi, mice were deeply anesthetized using isoflurane until unresponsive to pain stimuli (toe pinch) followed by cervical dislocation. The whole brain was harvested, and the hippocampus and subventricular zone (SVZ) were removed under a dissection microscope. These brain regions were then processed to obtain a single cell-suspension. Briefly, the hippocampi and SVZ were incubated for 20 mins at 37°C in an enzyme mixture containing 15 U/ml papain (Sigma Aldrich cat. no. P3125), 1 U/ml dispase (Gibco Life Technologies cat. no. 17105–041), 5 mM L-cysteine (Sigma Aldrich cat. no. 168149) and 1 mM of DNase (Roche cat. no. 1010415900115). The tissue was mechanically dissociated by pipetting 15–20 times in 500 µL of Dulbecco’s Modification of Eagle’s Medium (DMEM; Corning cat. no. 50–013-PC) and incubated for 10 mins to allow for undissociated tissue to settle at the bottom. Then, 400 µL of the supernatant were collected and again subjected to mechanical dissociation in an additional 400 µL of DMEM. After allowing the sample to stand for 10 mins, 700 µL of the supernatant were collected and centrifuged to obtain a pellet of single cell isolates. The single cell isolates were then fixed with Cytoperm/Cytofix solution (BD Biosciences cat. no. 554722) for 30 mins at 4°C in the dark. The fixed cells were stained with the following primary antibodies in perm wash (BD Biosciences cat. no. 554723) for 1 hour at 4°C: Rabbit anti-β-III tubulin (Cell Signaling Technology cat. no. 5568; 1;50), Alexa Fluor 647-Nestin (BD Biosciences cat. no. 560393; 1:1), Alexa Flour 488-O4 (R&D systems cat. no. FAB1326G; 1:6), APC-A2B5 (Miltenyi Biotec cat. no. 130-123-800; 1:10), rabbit anti-GFAP (DAKO cat. no. Z0334; 1:50), mouse anti-PDGFRα (Life Technologies cat. no. 14140182; 1:50), goat anti-Olig2 (R&D systems cat. no. AF2418; 1:25), and rabbit anti-TPPPp25 (Abcam cat. no. ab92305; 1:50). After one hour, the primary antibody was discarded, and the samples were incubated in the following secondary antibodies for one hour at 4°C: Alexa Fluor 488-donkey anti-mouse (Life Technologies #A21202; 1:1000), Alexa Fluor 488 goat anti-rabbit (Life Technologies cat. no. A11008, 1:1000), PE-goat anti-rabbit IgG (eBiosciences cat. no. 12473981; 1:100 or 1:200), Alexa Fluor 680-donkey anti-goat (Life Technologies cat. no. A21084; 1:200), and goat anti-rabbit PECy5.5 (Life Technologies cat. no. L42018; 1:200). Cells were then analyzed on an Attune NxT flow cytometer (Life Technologies cat. no. A24863) using Attune Cytometric Software v5.3.0. For each panel of antibodies, 1x10 6 events were counted. Gating was based on Fluorescent Minus One (FMO) control, which were applied as described previously 46 , 47 . Bromodeoxyuridine (BrdU) labeling of O4 + cells At 6–8 dpi, mice were injected intraperitoneally with BrdU (BD Biosciences cat. no. 550891; 100mg/kg). At 9 dpi, the brains were harvested, and the hippocampi and the subventricular zones (SVZ) were dissected and processed for flow cytometry as described for neural cells. Before fixing the cells as detailed above, samples were incubated with DNAse I (1U/µl) (Thermo Fisher cat. no. EN0521) for 40 mins at 37°C. Post fixing, the cells were processed in the same manner as for neural cell flow. The primary antibodies used were Alexa Flour 488-anti-O4 (R&D systems cat. no. FAB1326G; 1:6), and anti-BrdU monoclonal Alexa Fluor 647 (Life Technologies cat. no. B35140; 1:50) for one hour on ice before being analyzed on the flow cytometer as previously mentioned. Statistical analysis All data was tested for normality using the Shapiro-Wilk test and was found to be normally distributed. An unpaired two-tailed Student’s t test was applied for all comparisons of MeV-infected and PBS-injected groups, and a p value lower than 0.050 was deemed significant. The Grubbs outlier test was used to identify and exclude statistical outliers. All statistical analysis was performed using GraphPad Prism software version 9 (GraphPad Software, Inc., La Jolla, CA). RESULTS Juvenile mice experience enduring disruptions in myelination after MeV infection To determine if developmental myelination is perturbed during a neuron-restricted viral infection, myelin proteins were quantified in juvenile CD46 + mice (10 days old) infected with MeV during acute infection (9 days post infection; dpi) and in survivors that reached adulthood (90 dpi; Fig. 1) 42 . PLP, one of the major myelin proteins, was measured in four brain regions by western blot analysis (Fig. 2A). Since PLP is expressed as two main isoforms, we quantified each isoform independently as a high or low molecular weight isoform (HMW and LMW) and both isoforms together as a measure of total PLP expression (Fig. 2B-I). At 9 dpi, PLP expression increased in the cerebellum, but declined in the cortex. By 90 dpi, PLP expression decreased in both the SVZ and hippocampus, with greater PLP expression in the cortex compared to PBS-injected controls. These results suggest that MeV infection disturbs PLP expression in a region-dependent manner, with an enduring loss in PLP after infection within the neurogenic niches (SVZ and hippocampus). We also quantified MBP, which is another major myelin protein, and two minor myelin proteins (myelin oligodendrocyte glycoprotein (MOG) and myelin associated glycoprotein (MAG)) by western blot analysis (Table 1 and Supplemental Fig. 1–2). Overall, loss of myelin protein expression was generally noted in the SVZ and hippocampus, particularly in the long-term survivors. In contrast, myelin protein expression increased in the cerebellum at 9 dpi and the cortex at 90 dpi, despite early loss of major myelin protein markers at 9 dpi in the latter structure. Together, these results show that myelin protein expression is disrupted in multiple brain regions after MeV infection, although the kinetics and extent of this disruption vary by brain region. Early loss of myelin lipids during a juvenile viral infection In addition to myelin-specific proteins, the myelin sheath is enriched in lipids such as cholesterol, glycosphingolipid, and galactosylceramides that are collectively needed for the insulation provided by the myelin sheath 18 . Because the myelin proteins were disturbed, we next measured the myelin lipids using FluoroMyelin Green (FMG) staining. FMG is a highly lipophilic dye that stains the lipid-rich myelin sheath more intensely than other cellular membranes and is widely used to quantify myelin in tissues 52 – 55 . Representative images of FMG staining in the hippocampus and corpus callosum are shown at 9 dpi (Fig. 3A-3D). Quantification of FMG staining confirmed a significant decrease in the FMG signal at 9 dpi (Fig. 3E-3G), but not at 90 dpi (Fig. 3H-3J). These results suggest that myelin lipids are decreased during acute MeV infection. When considered together with the disturbances in myelin proteins (Fig. 2, Supplemental Fig. 1, Supplemental Fig. 2, and Table 1), our findings show that a neuronal MeV-infection is detrimental to developmental myelination in the juvenile brain. Sustained expansion of the OPC pool following MeV infection As myelination was perturbed at both 9 and 90 dpi, we examined the status of the OPCs which are the cells that differentiate into mature OLs. We focused on the OPC pool in the hippocampus and the SVZ because these regions are rich in NSCs that can ultimately give rise to OPCs 56 . As NSCs also differentiate into astrocytes, we quantified immature glia by labeling for A2B5, which can mark both early astrocytes and OPCs, as well as mature astrocytes by labeling for GFAP 57 . OPCs were labeled with the oligodendrocyte marker O4 antibody (O4 + ) 33,58 . Each lineage marker was assessed individually by flow cytometry in both brain regions at 9 and 90 dpi (Fig. 4A). In the hippocampus, immature glia and astrocyte numbers did not change at 9 dpi (Fig. 4B and 4C, respectively); however, the OPC pool increased during MeV infection (Fig. 4D; p = 0.006). In the SVZ, we observed an increase in immature glial cells (Fig. 4E) and a trend towards an increase in OPCs (Fig. 4G; p = 0.0524), while astrocytes remained unchanged compared to mock-infected controls at 9 dpi (Fig. 4F). Because abnormal expression of myelin proteins persisted in the surviving mice (Table 1), we next determined OPC numbers in the hippocampus and SVZ at 90 dpi. In the hippocampus, immature glial cells (A2B5 + , Fig. 5A) and OPCs (O4 + , Fig. 5C) were increased with infection, while astrocytes were unaltered (Fig. 5B). In the SVZ, the OPC pool increased with infection (Fig. 5F), while both the immature glia and astrocytes remained unchanged at 90 dpi (Fig. 5D and 5E, respectively). Together, these results suggest that the OPC pool undergoes long-term expansion in the hippocampus and SVZ after MeV infection in juvenile mice. OPC proliferation remains unchanged after MeV infection As the OPC pool increased at both 9 dpi (Fig. 4) and 90 dpi (Fig. 5), we next sought to determine the source of the new OPCs. In the postnatal brain, OPCs are one of the most proliferative cell types 59 . Therefore, we hypothesized that OPC proliferation may account for the increased number of OPCs during infection. To quantify OPC proliferation, we determined the percentage of proliferating OPCs through bromodeoxyuridine (BrdU) labeling to mark cells actively synthesizing DNA 60 . BrdU was injected daily from 6–8 dpi to capture the period before 9 dpi, where we first observed an increase in OPCs (Fig. 6A). The total population of BrdU + cells and cells that co-labeled for BrdU and O4 were measured to assess all proliferating cells and proliferating OPCs, respectively (Fig. 6B). Although substantial expansion of OPC pool was detected in MeV-infected groups, both BrdU + cells and BrdU + O4 + cells were unaltered with infection in the hippocampus (Fig. 6C and 6D) and the SVZ (Fig. 6E and 6F). These results indicate that the expansion in the OPC pool at 9 dpi (Fig. 4) may not be attributed to OPC proliferation per se . NSCs differentiate to form OPCs in both neurogenic niches during a juvenile CNS infection Another potential source of OPCs in the brain is differentiation of NSCs in the neurogenic niches 56 , 57 . We previously observed that NSC numbers transiently decline during acute infection (5 and 9 dpi), which was attributed only in part to modest cell death and differentiation into immature neurons 42 . Thus, we asked if the decrease in the NSC pool could also be explained by increased commitment to the OPC lineage. For these studies, we measured cells that co-labelled for OPC and NSC markers (O4 and nestin, respectively) at 5 dpi to capture events prior to the OPC expansion and myelin disturbances at 9 dpi (Fig. 7A). Using flow cytometry, we analyzed O4 + cells within the NSC population and the total number of cells co-labeling with nestin and O4 (Fig. 7B). Within the NSC pool (nestin + cells), greater numbers of cells expressed O4 in the hippocampus by 5 dpi (Fig. 7C) and 9 dpi (Fig. 7E), suggesting that more NSCs are being driven to differentiate to OPCs. Thus, the total number of cells that express both nestin and O4 (nestin + /O4 + ) increased at 5 dpi in the hippocampus (Fig. 7D). However, we did not observe an increase in the total nestin + /O4 + cells at 9 dpi (Fig. 7F), perhaps because the total nestin + population is reduced at this time point 42 . Similarly, in the SVZ, O4 + cells within the NSC pool increased at 5 dpi (Fig. 7G) and thus the total number of nestin + /O4 + cells also increased (Fig. 7H). At 9 dpi, we did not observe an increase in O4 expression within the NSC pool in the SVZ (Fig. 7I) and the total numbers decreased (Fig. 7J), which can be attributed to the decrease in total nestin + cells 42 . We also observed that the total number of O4 + cells does not change at 5 dpi but is increased by 9 dpi in the infected mice (Supplemental Fig. 3). Together, these results suggest that NSCs differentiate to form OPCs in both the hippocampus and SVZ as early as 5 dpi, which may eventually contribute to expansion of the OPC pool by 9 dpi. Continued maturation into the oligodendrocyte lineage in the brains of surviving mice Since the OPC pool increased in the surviving mice—despite abnormal myelination—we considered whether the OPCs failed to mature into OLs, which are ultimately responsible for myelination 61 . OPCs differentiate into OLs through a multistep process by forming early OPCs, intermediate OPCs, late OPCs, and finally mature OLs 58 . We utilized flow cytometry to quantify each of these stages using a series of OL markers (Table 2). For the gating strategy, Olig2, a pan oligodendrocyte marker, was used as the main gate to capture all cells in the different stages of OL differentiation (marked by a black box, Fig. 8A) 62 , 63 . In both the hippocampus and the SVZ, the total number of Olig2 + cells did not change with infection (data not shown), suggesting either that there is not an appreciable loss of OLs or that there is a drive to replace lost OLs during infection, resulting in a stable number of Olig2 + cells. When we examined the stages of OL differentiation in the hippocampus, we observed that early OPCs (Olig2 + /PDGFRα + /A2B5 − ; Fig. 8B) and intermediate OPCs (Olig2 + O4 + PDGFRα − A2B5 − ; Fig. 8C) remained unchanged whereas late OPCs (Olig2 + /O4 + /Tppp-p25 + /PDGFRα − /A2B5 − ; Fig. 8D), and OLs (Olig2 + /Tppp-p25 + /O4 − /PDGFRα − /A2B5 − ; Fig. 8E) increased in the surviving mice in adulthood (90 dpi). In the SVZ, we did not observe changes in the early OPCs (Fig. 8F), intermediate OPCs (Fig. 8G), and late OPCs (Fig. 8H). However, mature OLs (Fig. 8I) increased at 90 dpi in the SVZ of the surviving mice. Collectively, these data show the presence of greater OLs in both the neurogenic niches, thus showing that OPCs are propelled towards mature phenotype in surviving mice, despite the loss of myelin proteins. Neuronal numbers remain constant despite a loss of axonal and synaptic markers after MeV infection Because we observed disturbances in myelin proteins and OPC/OL numbers, we tested whether these disruptions were accompanied by a loss of neurons, which depend upon proper myelination for survival 64 , 65 . Using β-III tubulin as a marker, we quantified the number of mature neurons at 9, 30, and 90 dpi in the hippocampus by flow cytometry. Neuronal numbers were unchanged during infection (Fig. 9A-9C), despite the eventual decline in myelin markers in the hippocampus. Given that neuronal numbers were maintained, we next questioned if the maturation of neurons was compromised during infection, given that neurons are infected in our model 41 . To address this question, we first investigated the expression of neuronal proteins that connect the axon to the myelin sheath, which are important for maintaining neuron-OL crosstalk and establishing the borders of the node and paranodes. Specifically, we measured contactin associated protein (Caspr), which tethers the myelin sheath at the paranode, and neurofascin-186 (NF186), which maintains the node between myelin sheaths 66 – 69 . We observed that although neuronal numbers were stable, the expression of both NF186 (Fig. 9D) and Caspr (Fig. 9E) significantly decreased in the infected mice at 90 dpi. These results suggest that neuronal-OL crosstalk may be compromised after infection at 90 dpi. Demyelination in the hippocampus is associated with decreased synaptic density and a loss of synaptic marker expression in postmortem MS brains 70 . Thus, we examined expression of the presynaptic proteins Synapsin-1 and − 2, which are involved in synapse formation and synaptic vesicle regulation 71 – 75 . We observed a significant reduction in both synapsin-1 isoforms (1a/1b; Fig. 9F); whereas synapsin-2a trended toward a decline (Fig. 9F, p = 0.0732) at 90 dpi. Together, our results indicate that even though neuronal numbers are stable after MeV infection, neuronal markers of appropriate myelination and synaptic function are reduced in surviving mice. DISCUSSION In this paper, we found that juvenile mice experience enduring disruptions in myelination during a persistent MeV infection, despite a robust expansion of the OPC pool. OPCs are a highly proliferative cell type; however, we found that the expanding OPC pool arose from differentiation of neural stem cells (NSCs) into OPCs in the neurogenic niches. This sustained production of new OPCs may indicate an effort to differentiate into mature OLs to rescue myelination in the surviving hosts, as supported by increased maturation markers in OL lineage cells in infected mice. Nevertheless, surviving mice exhibit abnormal nodal and synaptic protein expression in the hippocampus, suggesting long-term damage to both neurons and myelin after acute MeV infection. Our results are consistent with findings in adult models of multiple sclerosis (MS), where OPCs are mobilized and attempt to differentiate, but remyelination is unsuccessful 35 , 76 – 78 . For decades, it has been speculated that viruses may act as a risk factor for demyelinating disease in susceptible hosts 39 , 79 , 80 . Recent studies have established Epstein Barr virus (EBV), a nearly ubiquitous herpesvirus, as a significant environmental risk factor in MS, with molecular mimicry between EBV proteins and myelin antigens (e.g., Glial-CAM) proposed as a mechanism for demyelination 81 , 82 . Virus-induced demyelination also occurs in more acute settings (e.g. encephalomyelitis), often involving immune-mediated mechanisms, including cytolysis of infected brain cells and/or production of myelin-specific antibodies or T cells. We now appreciate that even mild peripheral infections can disturb myelination through the induction of cytokines and chemokines, such as in respiratory SARS-COV2 infection in mice 83 . Although we do not yet know the immune factors that contribute to demyelination in this study, our findings suggest a slow, chronic myelin disruption in the hippocampus after MeV infection in young mice. The progressive nature of demyelination in our study bears similarities with Theiler’s murine encephalomyelitis virus (TMEV), a mouse pathogen that has been well-studied as a model of demyelinating disease. With neuroattenuated strains of TMEV, neurons are infected first during the acute infection, followed a month later by a chronic demyelinating phase with persistent infection in OLs and other glial cells 84 – 86 . Chronic demyelination by TMEV requires the establishment of persistent infection and results in part from immunopathology against the infected OLs. We suspect that the persistent nature of MeV infection in neurons similarly creates an inflammatory environment that leads to demyelination in the CD46 + mice. However, a key difference in our study is that MeV remains restricted to neurons in the juvenile CD46 + mice, which may suggest a more indirect role for the immune response in our model. Our findings further show that demyelination can occur in the absence of direct OL infection by a virus in the juvenile brain. Myelination and remyelination are highly dependent on neuronal activity; neurons that are electrically silenced receive poor myelination compared to electrically-active counterparts 64 , 65 . Thus, in our model, an alternative explanation for disrupted myelination could be that the neurons themselves are damaged, perhaps due to infection by MeV or exposure to inflammatory mediators. During acute MeV infection, juvenile CD46 + mice express multiple cytokines (e.g., interferon-gamma (IFNγ) and TNFα) that can modulate neuronal signaling 42 . IFNγ can increase inhibitory tone through release of GABA, whereas TNFα can increase neuronal excitability. In addition, MeV infection of rat cortical neurons in vitro has been shown to decrease voltage-gated Ca2 + currents 87 . One can therefore speculate that the balance of inflammatory cytokines during viral infection transiently dampens neuronal signaling and thereby deters engagement with myelinating OLs. Although we did not determine the electrical conductivity of neurons in this study, the reduction in pre-synaptic and nodal markers in surviving mice is suggestive of disturbances in neuronal signaling, which may ultimately contribute to diminished myelination or remyelination by neighboring OLs. We show that myelin disturbances in the neurogenic niches occur in conjunction with an expansion of the OPC pool, suggestive of an effort to repair myelin damage in the brain. A similar increase in NG2 + OPCs has also been reported in an encephalogenic model of TMEV infection, where NG2 + cell numbers and reactivity increased with infection in the hippocampi of adult mice 88 . In our model, we also found that the NSCs were contributing to the pool of O4 + cells. This phenomenon of “fate switching” of NSCs to OPCs is seen in models of MS, other demyelinating models, and in the presence of stress hormones such as glucocorticoids 89 – 91 . Although the exact molecular trigger is not yet clear, we speculate that a similar phenomenon is occurring in our model, where the stresses of the infection and inflammation induce NSC differentiation into OPCs. Intriguingly, studies in older mice suggest that OLs derived from NSCs may possess greater remyelination capacity than OLs derived directly from endogenous OPCs 92 . In a model of cuprizone-induced demyelination, NSCs in the SVZ contributed to the formation of new OLs and were essential for protection against axonal loss 93 . Thus, the generation of OPCs from the NSC pool may represent a more effective strategy for preserving neurons during demyelination. In our study, the kinetics and extent of demyelination varied by brain region after MeV infection. Multiple variables could contribute to region-specific outcomes in the brain, including neuronal phenotypes that influence synaptic connectivity and, possibly, rates of viral uptake, the local viral load, cytokine levels, immune cell infiltration, or the stage of myelination. For instance, PLP levels increased in the cerebellum during acute infection, but resolved by 90 dpi; whereas PLP levels declined in the cortex at 9 dpi and then increased at 90 dpi. Developmental myelination generally follows a posterior to anterior pattern, where myelination is initiated and completed early in the cerebellum and relatively late in the association cortex. Thus, the stage of developmental myelination may influence the response to inflammatory stimuli or infection. In support of this possibility, influenza A infection of neonatal mice (5 days old) is similarly associated with greater MBP expression in the cerebellum at 21 dpi, which the authors interpreted as a response to hypomyelination 94 . Another explanation could be regional and temporal differences in viral load or cytokine expression after infection. Here, we demonstrated that the SVZ had a more rapid and profound loss of myelin proteins than the hippocampus. In our prior studies, the SVZ also had more MeV-infected neurons and expressed higher levels of inflammatory cytokines during acute infection than the hippocampus, which suggests that degree of demyelination may be tuned by the local level of inflammation 42 . A limitation of our study is that we cannot yet discern whether the loss of myelin markers is due a blockade in developmental myelination (e.g., the initial myelin sheath is not produced properly) or due to the destruction of existing myelin. Given the age of the juvenile mice at infection (10 days old), it is feasible that both mechanisms are in play. During acute infection, IFNγ is highly expressed and is required for viral control in CD46 + mice 42 , 95 . However, IFNγ has also been shown to inhibit developmental myelination by blocking OPC proliferation and maturation 96 . In the CD46 + mice that survive to adulthood, we speculate that an autoimmune reaction may develop over time as the persistent infection is slowly resolved. Both innate immune cells (e.g., macrophage/microglia, neutrophils, and natural killer (NK) cells) and adaptive immune cells (CD4 and CD8 T cells, B cells) infiltrate the brain at 9 dpi, and B cells are still detected at 90 dpi, suggesting a sustained immune response 42 . Importantly, previous studies using a rodent-adapted MeV strain in rats concluded that a subacute measles encephalomyelitis (SAME) occurs after intracerebral MeV inoculation primarily due to a permissive inflammatory environment by the infection. The authors further add that this inflammatory microenvironment thus provides a conducible milieu for an autoimmune reaction to develop against myelin 97 . Thus, we speculate that myelination is disrupted at multiple levels post MeV-infection. Collectively, our study highlights that CNS viral infections during early life can have long-lasting cellular and developmental consequences even when survivors reach adulthood. We show that even if the virus infects only neurons 41 , 43 , 44 , the function of other brain cells can be indirectly impaired with lasting consequences for brain development. Importantly, even though demyelination was detected in multiple regions of the brain at 90 dpi, we did not note overt symptoms or neurological signs at this time point. However, over time, some of these surviving mice develop abnormal gait, partial paralysis, and seizures by 150 dpi when we found MeV to be undetectable 42 , which suggests that efforts to repair or restore myelination are ultimately unsuccessful. Future studies are needed to determine the extent of behavioral and motor impairments and the quality of myelination in long-term survivors. Ultimately, our studies reveal that survival after a childhood infection does not guarantee benign cellular outcomes. Abbreviations CNS: Central nervous system ADEM: Acute Disseminated Encephalomyelitis OL: Oligodendrocyte MBP: Myelin basic protein PLP: Myelin proteolipid protein P10: Postnatal day 10 OPCs: Oligodendrocyte progenitor cells NSCs: Neural stem cells MS: Multiple sclerosis MeV: Measles virus SSPE: Subacute sclerosing panencephalitis MAG: Myelin associated glycoprotein NSE: Neuron specific enolase dpi: Days post infection HMW: High molecular weight LMW: Low molecular weight SVZ: Sub-ventricular zone MOG: Myelin oligodendrocyte protein FMG: FluoroMyelin Green BrdU: Bromodeoxyuridine Caspr: Contactin associated protein Declarations Ethics approval : All animal studies were approved by the Duquesne University Institutional Animal Care and Use Committee (Protocol #1809-08 and #1865). Consent for publication : Not applicable. Availability of data and materials : The datasets used during the current study are available from the corresponding author upon reasonable request. Competing interests : The authors declare that they have no competing interests. Funding : This work was supported by grants from the NIH, the State of Pennsylvania CURE Fund, and the Charles Henry Leach, III Foundation. Authors’ ’contributions : YK performed the data collection, analysis, and prepared the figures. MC contributed to data collection in figures 2-6 and 8. CP, NL, and KY acquired data in figure 2 and Table 1. VS and KY assisted with mouse work in figures 2-3 and 8. AF acquired data in Figure 8. RKL contributed to statistical analyses and figures 2-3. YK and LO wrote the main manuscript. All authors reviewed and approved the final manuscript. References Raper J, et al. Long-term alterations in brain and behavior after postnatal Zika virus infection in infant macaques. 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Experimental measles encephalomyelitis in the rat: Generation of measles virus (MV) and T-lymphocyte cell lines specific for myelin basic protein (MBP). in Verhandlungen der Deutschen Gesellschaft für Neurologie 472–473Springer Berlin Heidelberg, Berlin, Heidelberg, (1987). Table 1 and 2 Table 1, 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementalFiles12212025.pptx SubmissionTables.pptx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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13:03:19","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":168630,"visible":true,"origin":"","legend":"","description":"","filename":"b94fae3b0d084717860ef41c52c00df21structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/ff0ab36a1deb7a3d1ccf9d53.xml"},{"id":100406908,"identity":"0d5a8b38-5527-4cbe-9244-e553e121c640","added_by":"auto","created_at":"2026-01-16 13:03:31","extension":"html","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182303,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/ac77ff438d6392169da1b9dc.html"},{"id":100406975,"identity":"5184dbb5-0d0f-4451-bce2-6a4b9499cbaf","added_by":"auto","created_at":"2026-01-16 13:03:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":157623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eJuvenile mouse model for neurotropic MeV infection:\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e Juvenile mice (postnatal day 10; P10) infected with measles virus (MeV; 2x10\u003csup\u003e4\u003c/sup\u003e PFU IC) or PBS were monitored daily for sickness and mortality for up to 90 days post infection (dpi). Symptoms such as hunched posture, seizures, and abnormal gait occur between 6-15 dpi (yellow bar). Approximately 25% of MeV-infected mice succumb to the infection by 15 dpi, while the remaining 75% survive to adulthood (90 dpi). Based on these observations, we harvested brains at 9 dpi to capture a time point during acute infection when symptoms are evident and at 90 dpi from survivors that reached adulthood.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/c4edbad3fdb90744128a92e1.png"},{"id":100406856,"identity":"d244aef1-57a5-4aa8-be53-918cc879bd8f","added_by":"auto","created_at":"2026-01-16 13:03:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":690950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003ePLP expression decreased in the brain after juvenile MeV infection:\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e\u003cem\u003e \u003c/em\u003eJuvenile mice were infected with measles virus (MeV), or PBS and brain tissue was harvested at 9- or 90-days post infection (dpi). The sub-ventricular zone (SVZ), hippocampus (Hippo), cerebellum (cere), and cortex were dissected and lysed for western blot analysis for myelin proteolipid protein (PLP). (A) Representative blots are shown for PLP in the SVZ at 9 dpi and 90 dpi. The high molecular weight (HMW) and low molecular weight (LMW) isoforms of PLP (green) and a loading control (GAPDH; red) are indicated for the PBS-injected and MeV-infected groups. Quantification of PLP at 9 dpi (left column) and 90 dpi (right column) are shown for the SVZ (B and C), the hippocampus (D and E), cerebellum (F and G), and cortex (H and I). All graphs from B-I were normalized to GAPDH of their respective blots and are presented as arbitrary units (A.U.). Statistical analysis was applied by unpaired two-tailed student t-test where *p\u0026lt;0.05 , **p\u0026lt;0.01, and ***p\u0026lt;0.001 (n=6-7 mice from 2-3 litters).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/b140217b11a95f5d88dc70d2.png"},{"id":100406776,"identity":"b7bae994-0bcf-4045-ad9c-5478113c4d15","added_by":"auto","created_at":"2026-01-16 13:03:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1233823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eDisruption in myelin lipids after juvenile MeV infection:\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e\u003cem\u003e \u003c/em\u003eFluorescent microscopy images of the hippocampus and corpus callosum in PBS-injected (A, B) and MeV-infected (C, D) juvenile mice at 9 dpi. Brain tissue was stained with Fluoromyelin Green (FMG). Images are captured using the same intensity and exposure times with no changes to the gain. The region of interest (dotted squares; A, C) mark the posterior end of the corpus callosum, which is magnified in panels B and D. Panels E-J represent images and data captured on an Odyssey imager. Whole brain representative images are shown for the FMG stain at 9 dpi (E. PBS and F. MeV) and 90 dpi (H. PBS and I. MeV). The dotted square in E represents the area including the hippocampus and the corpus callosum, which was traced by hand on each slice and quantified for FMG signal. Data were represented as FMG signal/area for 9 dpi (G) and 90 dpi (J). 10-20 slices per animal were traced and averaged to obtain the FMG quantification in G and J. Statistical analysis was applied by unpaired two-tailed student t-test where ****p\u0026lt;0.0001 (n = 4 mice/condition).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/c17290659b33794f31a57a35.png"},{"id":100407045,"identity":"9fdfa051-0f93-427c-8406-bf12c04c6473","added_by":"auto","created_at":"2026-01-16 13:03:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":201041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eThe oligodendrocyte progenitor cell (OPC) pool expands after juvenile MeV infection: \u003c/strong\u003e\u003c/em\u003e\u003c/u\u003eJuvenile mice were infected with measles virus (MeV), or PBS and their brain was harvested at 9 days post infection (dpi) for flow cytometry. Panel A depicts the gating strategy that was applied, where each marker was gated for separately. A2B5, GFAP, and O4 was used to label immature glia, astrocytes, and oligodendrocyte progenitor cells (OPCs), respectively. A2B5\u003csup\u003e+ \u003c/sup\u003e(B and E), GFAP\u003csup\u003e+ \u003c/sup\u003e(C and F), and O4\u003csup\u003e+ \u003c/sup\u003e(D and G) cells are shown for the hippocampus (middle row; B-D) and sub-ventricular zone (SVZ) (bottom row; E-G). Statistical analysis was applied by unpaired two-tailed student t-test where *p\u0026lt;0.05 and ***p\u0026lt;0.001 and n = 17-19 mice.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/750282b5513c45ef66cdaf96.png"},{"id":100406233,"identity":"fc6cc602-aec5-4a10-af83-1b8bfdc91cee","added_by":"auto","created_at":"2026-01-16 12:58:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":228137,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eLong-term expansion of oligodendrocyte progenitor cells (OPCs) after juvenile MeV infection\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e: Juvenile mice were infected with measles virus (MeV), or PBS and their brain was harvested at 90 days post infection (dpi). A2B5, GFAP, and O4 was used to label immature glia, astrocytes, and OPCs respectively at 90 dpi via flow cytometry. A2B5\u003csup\u003e+ \u003c/sup\u003e(A and D), GFAP\u003csup\u003e+ \u003c/sup\u003e(B and E), and O4\u003csup\u003e+ \u003c/sup\u003e(C and F) cell numbers are shown for the hippocampus (upper panel; A-C) and sub-ventricular zone (SVZ) (lower panel; D-F). Statistical analysis was applied by unpaired two-tailed student t-test where *p\u0026lt;0.05 and ****p\u0026lt;0.0001 and n = 26-27.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/92b17d366854fbc137b296c0.png"},{"id":100405905,"identity":"82fe4e62-9c26-4680-ad55-df26380c3048","added_by":"auto","created_at":"2026-01-16 12:27:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":289613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eOPC proliferation remains unaltered after juvenile MeV infection\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e\u003cem\u003e\u003cstrong\u003e: \u003c/strong\u003e\u003c/em\u003eJuvenile mice were infected with measles virus (MeV), or PBS at postnatal day 10 (P10) followed by BrdU (Bromodeoxyuridine; 100mg/kg) intraperitoneally (IP) injections at 6 days post infection (dpi), 7 dpi, and 8 dpi. Their brain was harvested at 9 dpi (A). Panel B shows the gating strategy used for data in C-F. BrdU and O4 were used to mark proliferating cells and oligodendrocyte progenitor cells (OPCs) respectively. BrdU\u003csup\u003e+ \u003c/sup\u003eand BrdU\u003csup\u003e+\u003c/sup\u003e O4\u003csup\u003e+\u003c/sup\u003e of total cells in the hippocampus (C and D) and the sub-ventricular zone (SVZ) (E and F) are shown at 9 dpi. Statistical analysis was applied by unpaired two-tailed student t-test where and n = 10-13.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/9a9925322d9b2f70d3440825.png"},{"id":100405897,"identity":"a8dfeab5-2d97-4417-87e4-54f042511155","added_by":"auto","created_at":"2026-01-16 12:27:32","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":377738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eNSCs move towards the OPC lineage as early as 5 days post infection:\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e\u003cem\u003e \u003c/em\u003eJuvenile mice were infected with measles virus (MeV) or PBS, and their brain was harvested either at 5 days post infection (dpi) or 9 dpi (A). Gating strategy used for these experiments is shown (B). O4 and Nestin were used to mark oligodendrocyte progenitor cells (OPCs) and neural stem cells (NSCs) respectively. O4\u003csup\u003e+ \u003c/sup\u003ecells within the Nestin\u003csup\u003e+\u003c/sup\u003e population and the O4\u003csup\u003e+\u003c/sup\u003e Nestin\u003csup\u003e+\u003c/sup\u003e pool of the total cells is shown in the hippocampus at 5 dpi (C and D) and at 9 dpi (E and F). Similarly, the O4\u003csup\u003e+\u003c/sup\u003e cells within the Nestin\u003csup\u003e+\u003c/sup\u003e population and the O4\u003csup\u003e+\u003c/sup\u003e Nestin\u003csup\u003e+\u003c/sup\u003e pool of the total cells is shown for the sub-ventricular zone (SVZ) at 5 dpi (G and H) and at 9 dpi (I and J). Statistical analysis was applied by unpaired two-tailed student t-test where *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, and ****p\u0026lt;0.0001; and n = 5-13 mice.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/eb5b6c10bc73ed7f4db6fd4f.png"},{"id":100421694,"identity":"d1c2a8cb-97cc-4573-9f30-73ffebd25be5","added_by":"auto","created_at":"2026-01-16 13:44:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":987959,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eExpansion of oligodendrocytes at different stages of differentiation after MeV infection at the juvenile age:\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e Juvenile mice were infected with measles virus (MeV), or PBS and their brain was harvested at 90 days post infection (dpi). PDGFRα, A2B5, Olig2, Tppp-p25, and O4 were used to label oligodendrocytes at different stages of differentiation. (A) Shows the gating strategy where Olig2+ cells (shown in black box) was used as the main gate to mark early oligodendrocyte progenitor cells (OPCs) while Olig2+ A2B5- PDGFRα- cells (outlined by a green box) was used to mark intermediate OPCs, late OPCs, and mature oligodendrocytes in the hippocampus and sub-ventricular zone (SVZ). The early OPCs (B), intermediate OPCs (C), late OPCs (D), and mature oligodendrocytes (E) are shown for hippocampus at 90 dpi. Similarly, early OPCs (F), intermediate OPCs (G), late OPCs (H), and mature oligodendrocytes (I) are shown for the SVZ at 90 dpi. Statistical analysis was applied by unpaired two-tailed student t-test where *p\u0026lt;0.05, **p\u0026lt;0.01, and ****p\u0026lt;0.0001 and n = 19-22 mice.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/400492856e18c2df7c629044.png"},{"id":100406493,"identity":"9507c1a0-f4d8-49d0-a14e-3c91217eee4a","added_by":"auto","created_at":"2026-01-16 13:02:38","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":799764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cu\u003e\u003cem\u003e\u003cstrong\u003eSurviving mice show long-term reductions in neurofascin186, caspr, and synapsin without hippocampal mature neuron loss\u003c/strong\u003e\u003c/em\u003e\u003c/u\u003e\u003cem\u003e\u003cstrong\u003e: \u003c/strong\u003e\u003c/em\u003eJuvenile mice were infected with measles virus (MeV), or PBS and their brain was harvested at 9-, 30-, and 90-days post infection (dpi). The hippocampus was dissected and processed for flow cytometry (A-C) and western blot (D-F). β-III tubulin was used to label mature neurons by flow cytometry as shown in the hippocampus at 9 dpi (A), 30 dpi (B), and 90 dpi (C). Western blot image and quantification of neurofascin186 (green) (D), Caspr (green) (F), and synapsin1/2 (green) (E) normalized by loading control, GAPDH of the respective blot (red) (D-F) is shown for the hippocampus at 90 dpi. Data is presented as arbitrary units (A.U.). Statistical analysis was applied by unpaired two-tailed student t-test where *p\u0026lt;0.05, **p\u0026lt;0.01, and n= 10-31 (A-C) and n=6-8 for (D-F).\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/36d0c9def4094dc0f85a1c60.png"},{"id":100998721,"identity":"fd4c842a-3423-492c-b887-780139c09c19","added_by":"auto","created_at":"2026-01-23 15:56:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6470898,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/04c009a5-dfdd-4d44-ae37-cf0ef371eff4.pdf"},{"id":100406018,"identity":"63c90dfb-28ad-4a38-bb44-bdc6e9269179","added_by":"auto","created_at":"2026-01-16 12:33:30","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7490536,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFiles12212025.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/5fe0e45e11dcdeb7e2a7ebce.pptx"},{"id":100406462,"identity":"b9d4ab5c-38a4-400c-96d0-3085df28c57b","added_by":"auto","created_at":"2026-01-16 13:02:28","extension":"pptx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1822556,"visible":true,"origin":"","legend":"","description":"","filename":"SubmissionTables.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8563506/v1/2fed3ebdfeb643e6535b8b40.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Oligodendrocyte progenitor cells are promoted in the neurogenic niches of the juvenile mouse brain during viral infection","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eViral infections of the central nervous system (CNS) are important causes of demyelination, encephalitis, and enduring neurological sequelae in children\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although substantial brain development occurs \u003cem\u003ein utero\u003c/em\u003e, the brain undergoes extensive maturation in childhood, with myelination, synaptogenesis, and synaptic pruning occurring throughout adolescence and even into adulthood \u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Viruses can disrupt these processes through direct infection of developing brain cells (\u003cem\u003ee.g.\u003c/em\u003e, neurons, oligodendrocytes) and/or through bystander damage by the ensuing immune response against the virus. Additionally, in young children (\u0026lt;\u0026thinsp;10 years), viral infections often precede the onset of acute disseminated encephalomyelitis (ADEM), an immune-mediated demyelinating disorder triggered by many different viruses (\u003cem\u003ee.g\u003c/em\u003e., measles, coronaviruses, influenza) after peripheral infection\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Thus, the immune response plays a multifaceted role in the young brain, where it may simultaneously contain viral spread while disturbing ongoing myelination.\u003c/p\u003e \u003cp\u003eIn the CNS, the myelin sheath is produced by oligodendrocytes (OLs). Upon selection of a neuronal axon, the OL plasma membrane wraps around a segment of the axon in tight concentric layers to form the sheath. In comparison to other cellular membranes, the membranes in the myelin sheath are enriched in lipids and myelin-specific proteins (e.g., myelin basic protein (MBP) and proteolipid protein (PLP)) that help to compact and stabilize the concentric layers. Myelination insulates the axon to allow for the propagation of faster action potentials and provides metabolic support to the neuron, further supporting axonal structure and integrity \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In humans, myelination begins in the second trimester of gestation, peaks at 2\u0026ndash;3 years of age, and continues into adolescence and adulthood, with varying kinetics depending upon the anatomical site \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Brain regions involved in sensory and motor function are myelinated early in childhood, while neocortical regions involved in higher-order cognition are myelinated into adulthood. Mice follow a similar temporal pattern, with a wave of immature OLs emerging by postnatal day 10 (P10), a peak of myelination between 2\u0026ndash;4 weeks of age, and continuation into adulthood (2\u0026ndash;8 months old) \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Overall, myelination is crucial to facilitate connections in the developing brain and for rapid and synchronized transfer of information \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOL numbers increase dramatically in childhood as myelination peaks, but OLs do not typically divide\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Rather, new OLs are produced by specialized stem cells known as oligodendrocyte precursor cells (OPCs), which commit to the OL lineage through a carefully orchestrated series of cues during developmental myelination. OPCs can originate from the proliferation of existing OPCs or the commitment of neural stem cells (NSCs) to the glial lineage. Indeed, OPCs are among the most highly proliferative cells in the CNS, with greater OPC proliferation in white matter regions than grey matter regions prior to differentiation. OPCs can respond to injury throughout life by proliferating and migrating to sites of damage, promoting glial scar formation, and supporting neuronal signaling, among other roles\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Despite their versatility, new OPCs are impeded in their differentiation into OLs in several neurological diseases in adults (\u003cem\u003ee.g.\u003c/em\u003e multiple sclerosis (MS), stroke, schizophrenia)\u003csup\u003e\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. These demyelinating diseases have provided insights into the pathological responses of adult OPCs. However, in the developing brain, the role of OPCs is poorly understood in neuroinflammatory diseases, including in the context of viral infections and the ensuing immune response.\u003c/p\u003e \u003cp\u003eMeasles virus (MeV) is associated with a rare but diverse array of CNS complications in children, including demyelinating illnesses. MeV has been shown to directly infect OLs and neurons in subacute sclerosing panencephalitis (SSPE), which is a lethal, persistent infection by MeV \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In patients who succumbed to MeV encephalomyelitis, multiple myelin markers (e.g., MBP and myelin associated glycoprotein (MAG)) were reduced although MeV antigen was undetectable in the brain, suggesting an immune-mediated attack on the myelin sheath \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. These studies suggest that MeV can induce demyelinating disease through direct infection of brain cells or through neuroinflammation. However, the response of developing OPCs and OLs to viral inflammation in childhood, when myelination is at its peak, is unknown.\u003c/p\u003e \u003cp\u003eWe previously studied MeV neuropathogenesis at the juvenile age (postnatal day 10) using NSE-CD46 mice (CD46+). In CD46\u0026thinsp;+\u0026thinsp;mice, the neuron-specific enolase (NSE) promoter drives the expression of the human CD46 gene, which encodes one of three MeV receptors \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. In CD46\u0026thinsp;+\u0026thinsp;mice, only mature neurons are infected by MeV, while other neural cells are spared from direct viral infection. In juvenile CD46\u0026thinsp;+\u0026thinsp;mice, ~\u0026thinsp;25% of mice developed neurological symptoms (e.g., altered gait, ocular symptoms, seizures) by two weeks post MeV-infection and succumbed, despite robust infiltration of immune cells and cytokine expression. The surviving juvenile mice (~\u0026thinsp;75%) remained asymptomatic up to three months post-infection, but ultimately developed neurological symptoms by five months, indicating declining neurological function \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Surprisingly, in both symptomatic and asymptomatic juvenile mice, NSC numbers transiently declined during acute infection, which was attributed only in part to modest cell death and limited differentiation into immature neurons, suggesting that NSCs may be marshalled toward a glial fate during viral infection \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven the progressive neurological decline in mice that survived MeV infection as juveniles, the aim of this study was to determine if developmental myelination was disrupted due to infection. Because MeV is restricted to neurons in the CD46\u0026thinsp;+\u0026thinsp;model, OPCs/OLs are spared from direct infection, thus allowing us to examine the bystander responses of these cells to the microenvironment created by the antiviral immune response and virally-infected neurons \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Ultimately, we speculate that juvenile OPCs are mobilized in the inflammatory microenvironment in the brain, but cannot overcome disruptions in developmental myelination, leading to long-term neurological consequences even after resolution of the initial infection.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals and ethics statement\u003c/h2\u003e \u003cp\u003eCD46\u0026thinsp;+\u0026thinsp;mice were maintained and treated according to the \u003cem\u003eInstitutional Animal Care and Use Committee of Duquesne University\u003c/em\u003e under approved protocols and the \u003cem\u003eNIH Guide for the Care and Use of Laboratory Animals.\u003c/em\u003e The CD46\u0026thinsp;+\u0026thinsp;mice were maintained on a 12h/12h light/dark cycle under controlled temperature conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u0026deg;C) with free access to food and water. Paired mating cages were established to generate juvenile mice for all experiments. Both male and female mice were used for the experiments reported herein.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasles virus (MeV) infections\u003c/h3\u003e\n\u003cp\u003ePostnatal day 10 (P10) CD46\u0026thinsp;+\u0026thinsp;juvenile mice were infected with measles virus (MeV)-Edmonston strain obtained from ATCC (American Type Culture Collection; Cat No: VR-24). The virus was passaged three times and plaque-assayed in Vero cells. The inoculum was diluted in sterile phosphate buffered saline (PBS) and administered intracerebrally with a 1cc syringe (BD cat. no. 309659) and 27\u003csup\u003e1/2\u0026minus;gauge\u003c/sup\u003e needle (BD cat. no. 305109). On P10, mice were lightly anesthetized with isoflurane and injected with 20 \u0026micro;l of MeV (2x10\u003csup\u003e4\u003c/sup\u003e PFU) or with 20 \u0026micro;l of PBS as a negative control. The MeV-infected or PBS control mice were observed daily for signs of sickness and mortality, for up to 9- or 90-days post infection (dpi).\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern blotting\u003c/div\u003e \u003cp\u003eOn the indicated dpi, mice were deeply anesthetized with isoflurane inhalation until unresponsive to pain stimuli (toe pinch), followed by cervical dislocation. The hippocampus, subventricular zone (SVZ), cerebellum, and cortex of each mouse were collected and lysed in 1\u0026times; Cell Lysis Buffer (Cell Signaling Technology; cat. no. 9803) with 1\u0026times; Protease Inhibitor Cocktail (Sigma-Aldrich; cat. no. P8340) (20 \u0026micro;L lysis buffer per mg tissue). Brain lysates were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing. The protein concentration of each lysate was measured using the Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (ThermoFisher Scientific; cat. no. 23225) on a TECAN Infinite M1000 plate reader. For the hippocampus, cerebellum, and cortex samples, 20 \u0026micro;g of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For the SVZ, 10 \u0026micro;g of protein were used for 9 dpi samples, whereas 15 \u0026micro;g of protein were used for 90 dpi samples because the SVZ dissections yielded relatively small pieces of tissue. The gel was blotted onto Immobilon-FL PVDF Membrane (Millipore; cat. no. IPFL00010) for 35 mins (semi-dry transfer) and the membranes were blocked using either a 1:1 mixture of 1\u0026times; PBS/Tween-20 solution (Sigma-Aldrich; cat. no. P3563) and Odyssey blocking buffer (LICOR Biosciences; cat. no. 927\u0026ndash;40000) or undiluted Intercept (TBS) blocking buffer (LICOR Biosciences; cat. no. 927-60001) for 1 hour at room temperature. The membranes were treated with primary antibody diluted in the aforementioned blocking solution overnight at 4\u0026deg;C on a rocker. The membranes were washed thrice with PBS-Tween (10 mins/wash) and incubated for one hour at room temperature in secondary antibody solutions, including goat anti-rabbit 800 (LI-COR Biosciences; cat. no. 926\u0026ndash;32211; 1:10,000), goat anti-rat 800 (LI-COR Biosciences; cat. no. 926-32219, 1:10,000), donkey anti-rabbit 680 (LI-COR Biosciences; cat. no. 926-68073, 1:10,000), and donkey anti-mouse 680 (LI-COR Biosciences; cat. no. 926\u0026ndash;68072; 1:10,000). The membranes were then washed thrice (10 mins/wash) in PBS-Tween and imaged on the Odyssey Infrared Imaging System (LI-COR Biosciences; model: Classic and M). Primary antibodies used were as follows: mouse anti-GAPDH (Millipore cat. no. MAB374; 1:10,000), rabbit anti-neurofascin186 (Cell Signaling Technology; cat. no. 15034s; 1:1000), rabbit anti-synapsin1/2 (Synaptic System; cat. no. 106002; 1:1000), rabbit anti-Caspr (Cell Signaling Technology cat. no. 97736S, 1:1000), rat anti-myelin basic protein (MBP; Millipore cat. no. MAB386, 1:500), rabbit anti-myelin proteolipid protein (PLP; Cell Signaling Technology cat. no. 28702, 1:1000), rabbit anti-myelin oligodendrocyte glycoprotein (MOG; Cell Signaling Technology cat. no. 96457T, 1:1000), and rabbit anti-myelin associated glycoprotein (MAG; Cell Signaling Technology cat. no. 9043T, 1:1000).\u003c/p\u003e\n\u003ch3\u003eFluoroMyelin Green staining\u003c/h3\u003e\n\u003cp\u003eMice were lightly anesthetized with isoflurane either 9- or 90-days post infection, before intraperitoneal injection with 3.8% choral hydrate in PBS. This was followed by cardiac perfusion with ice-cold PBS followed by 4% paraformaldehyde/PBS (PFA/PBS; Affymetrix cat. no. 19943). Whole brains were collected, fixed with 4% PFA/PBS for 24 hours, and cryoprotected with 30% sucrose (Fisher Scientific cat. no. BP220\u0026ndash;212) in PBS at 4\u0026deg;C. Brains were then sectioned on a sliding/freezing microtome in the sagittal plane at 40\u0026micro;m thickness as a 1-in-4 free floating series and stored at -20\u0026deg;C in cryoprotectant \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. For staining brain slices with FluoroMyelin Green (ThermoFisher Scientific cat. no. F34651; 1:150), the manufacturer\u0026rsquo;s protocol was followed with minor modifications. Briefly, the tissue was washed in 10 mM PBS for 20 mins before staining them with FluoroMyelin green (1:150) for 20 mins at room temperature. The tissue sections were then washed three times for 10 mins in 10 mM PBS before mounting. ProLong Gold Antifade Mountant was used to mount FluoroMyelin-stained tissue (Thermo Fisher Scientific cat. no. P36930) on Superfrost plus slides (Fisher Scientific cat. no. 1255015).\u003c/p\u003e \u003cp\u003eThe areas of the hippocampus and corpus callosum were traced to quantify the FluoroMyelin green signal. The tissue was imaged on an Odyssey Infrared Imaging System (LI-COR Biosciences; Odyssey M model) at 5 \u0026micro;m resolution and on an epifluorescence microscope Olympus IX73 (B\u0026amp;B Microscopes). Tracings were performed using the Image Studio Lite Ver 5.2 (LI-COR Biosciences) to determine the total FluoroMyelin green signal, which was then expressed as a fraction of the trace area. PBS-injected controls and MeV infected animals were used (n\u0026thinsp;=\u0026thinsp;4) at both 9 and 90 dpi. The hippocampus and corpus callosum from 10\u0026ndash;20 slices per animal were traced after discarding torn slices.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry for neural cells\u003c/h3\u003e\n\u003cp\u003eOn the indicated dpi, mice were deeply anesthetized using isoflurane until unresponsive to pain stimuli (toe pinch) followed by cervical dislocation. The whole brain was harvested, and the hippocampus and subventricular zone (SVZ) were removed under a dissection microscope. These brain regions were then processed to obtain a single cell-suspension. Briefly, the hippocampi and SVZ were incubated for 20 mins at 37\u0026deg;C in an enzyme mixture containing 15 U/ml papain (Sigma Aldrich cat. no. P3125), 1 U/ml dispase (Gibco Life Technologies cat. no. 17105\u0026ndash;041), 5 mM L-cysteine (Sigma Aldrich cat. no. 168149) and 1 mM of DNase (Roche cat. no. 1010415900115). The tissue was mechanically dissociated by pipetting 15\u0026ndash;20 times in 500 \u0026micro;L of Dulbecco\u0026rsquo;s Modification of Eagle\u0026rsquo;s Medium (DMEM; Corning cat. no. 50\u0026ndash;013-PC) and incubated for 10 mins to allow for undissociated tissue to settle at the bottom. Then, 400 \u0026micro;L of the supernatant were collected and again subjected to mechanical dissociation in an additional 400 \u0026micro;L of DMEM. After allowing the sample to stand for 10 mins, 700 \u0026micro;L of the supernatant were collected and centrifuged to obtain a pellet of single cell isolates. The single cell isolates were then fixed with Cytoperm/Cytofix solution (BD Biosciences cat. no. 554722) for 30 mins at 4\u0026deg;C in the dark. The fixed cells were stained with the following primary antibodies in perm wash (BD Biosciences cat. no. 554723) for 1 hour at 4\u0026deg;C: Rabbit anti-β-III tubulin (Cell Signaling Technology cat. no. 5568; 1;50), Alexa Fluor 647-Nestin (BD Biosciences cat. no. 560393; 1:1), Alexa Flour 488-O4 (R\u0026amp;D systems cat. no. FAB1326G; 1:6), APC-A2B5 (Miltenyi Biotec cat. no. 130-123-800; 1:10), rabbit anti-GFAP (DAKO cat. no. Z0334; 1:50), mouse anti-PDGFRα (Life Technologies cat. no. 14140182; 1:50), goat anti-Olig2 (R\u0026amp;D systems cat. no. AF2418; 1:25), and rabbit anti-TPPPp25 (Abcam cat. no. ab92305; 1:50). After one hour, the primary antibody was discarded, and the samples were incubated in the following secondary antibodies for one hour at 4\u0026deg;C: Alexa Fluor 488-donkey anti-mouse (Life Technologies #A21202; 1:1000), Alexa Fluor 488 goat anti-rabbit (Life Technologies cat. no. A11008, 1:1000), PE-goat anti-rabbit IgG (eBiosciences cat. no. 12473981; 1:100 or 1:200), Alexa Fluor 680-donkey anti-goat (Life Technologies cat. no. A21084; 1:200), and goat anti-rabbit PECy5.5 (Life Technologies cat. no. L42018; 1:200). Cells were then analyzed on an Attune NxT flow cytometer (Life Technologies cat. no. A24863) using Attune Cytometric Software v5.3.0. For each panel of antibodies, 1x10\u003csup\u003e6\u003c/sup\u003e events were counted. Gating was based on Fluorescent Minus One (FMO) control, which were applied as described previously \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBromodeoxyuridine (BrdU) labeling of O4\u003csup\u003e+\u003c/sup\u003e cells\u003c/h2\u003e \u003cp\u003eAt 6\u0026ndash;8 dpi, mice were injected intraperitoneally with BrdU (BD Biosciences cat. no. 550891; 100mg/kg). At 9 dpi, the brains were harvested, and the hippocampi and the subventricular zones (SVZ) were dissected and processed for flow cytometry as described for neural cells. Before fixing the cells as detailed above, samples were incubated with DNAse I (1U/\u0026micro;l) (Thermo Fisher cat. no. EN0521) for 40 mins at 37\u0026deg;C. Post fixing, the cells were processed in the same manner as for neural cell flow. The primary antibodies used were Alexa Flour 488-anti-O4 (R\u0026amp;D systems cat. no. FAB1326G; 1:6), and anti-BrdU monoclonal Alexa Fluor 647 (Life Technologies cat. no. B35140; 1:50) for one hour on ice before being analyzed on the flow cytometer as previously mentioned.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data was tested for normality using the Shapiro-Wilk test and was found to be normally distributed. An unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test was applied for all comparisons of MeV-infected and PBS-injected groups, and a \u003cem\u003ep\u003c/em\u003e value lower than 0.050 was deemed significant. The Grubbs outlier test was used to identify and exclude statistical outliers. All statistical analysis was performed using GraphPad Prism software version 9 (GraphPad Software, Inc., La Jolla, CA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eJuvenile mice experience enduring disruptions in myelination after MeV infection\u003c/h2\u003e \u003cp\u003eTo determine if developmental myelination is perturbed during a neuron-restricted viral infection, myelin proteins were quantified in juvenile CD46\u0026thinsp;+\u0026thinsp;mice (10 days old) infected with MeV during acute infection (9 days post infection; dpi) and in survivors that reached adulthood (90 dpi; Fig.\u0026nbsp;1) \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. PLP, one of the major myelin proteins, was measured in four brain regions by western blot analysis (Fig.\u0026nbsp;2A). Since PLP is expressed as two main isoforms, we quantified each isoform independently as a high or low molecular weight isoform (HMW and LMW) and both isoforms together as a measure of total PLP expression (Fig.\u0026nbsp;2B-I). At 9 dpi, PLP expression increased in the cerebellum, but declined in the cortex. By 90 dpi, PLP expression decreased in both the SVZ and hippocampus, with greater PLP expression in the cortex compared to PBS-injected controls. These results suggest that MeV infection disturbs PLP expression in a region-dependent manner, with an enduring loss in PLP after infection within the neurogenic niches (SVZ and hippocampus).\u003c/p\u003e \u003cp\u003eWe also quantified MBP, which is another major myelin protein, and two minor myelin proteins (myelin oligodendrocyte glycoprotein (MOG) and myelin associated glycoprotein (MAG)) by western blot analysis (Table\u0026nbsp;1 and Supplemental Fig.\u0026nbsp;1\u0026ndash;2). Overall, loss of myelin protein expression was generally noted in the SVZ and hippocampus, particularly in the long-term survivors. In contrast, myelin protein expression increased in the cerebellum at 9 dpi and the cortex at 90 dpi, despite early loss of major myelin protein markers at 9 dpi in the latter structure. Together, these results show that myelin protein expression is disrupted in multiple brain regions after MeV infection, although the kinetics and extent of this disruption vary by brain region.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEarly loss of myelin lipids during a juvenile viral infection\u003c/h2\u003e \u003cp\u003eIn addition to myelin-specific proteins, the myelin sheath is enriched in lipids such as cholesterol, glycosphingolipid, and galactosylceramides that are collectively needed for the insulation provided by the myelin sheath \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Because the myelin proteins were disturbed, we next measured the myelin lipids using FluoroMyelin Green (FMG) staining. FMG is a highly lipophilic dye that stains the lipid-rich myelin sheath more intensely than other cellular membranes and is widely used to quantify myelin in tissues \u003csup\u003e\u003cspan additionalcitationids=\"CR53 CR54\" citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Representative images of FMG staining in the hippocampus and corpus callosum are shown at 9 dpi (Fig.\u0026nbsp;3A-3D). Quantification of FMG staining confirmed a significant decrease in the FMG signal at 9 dpi (Fig.\u0026nbsp;3E-3G), but not at 90 dpi (Fig.\u0026nbsp;3H-3J). These results suggest that myelin lipids are decreased during acute MeV infection. When considered together with the disturbances in myelin proteins (Fig.\u0026nbsp;2, Supplemental Fig.\u0026nbsp;1, Supplemental Fig.\u0026nbsp;2, and Table\u0026nbsp;1), our findings show that a neuronal MeV-infection is detrimental to developmental myelination in the juvenile brain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSustained expansion of the OPC pool following MeV infection\u003c/h2\u003e \u003cp\u003eAs myelination was perturbed at both 9 and 90 dpi, we examined the status of the OPCs which are the cells that differentiate into mature OLs. We focused on the OPC pool in the hippocampus and the SVZ because these regions are rich in NSCs that can ultimately give rise to OPCs \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. As NSCs also differentiate into astrocytes, we quantified immature glia by labeling for A2B5, which can mark both early astrocytes and OPCs, as well as mature astrocytes by labeling for GFAP \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. OPCs were labeled with the oligodendrocyte marker O4 antibody (O4\u003csup\u003e+\u003c/sup\u003e) \u003csup\u003e33,58\u003c/sup\u003e. Each lineage marker was assessed individually by flow cytometry in both brain regions at 9 and 90 dpi (Fig.\u0026nbsp;4A). In the hippocampus, immature glia and astrocyte numbers did not change at 9 dpi (Fig.\u0026nbsp;4B and 4C, respectively); however, the OPC pool increased during MeV infection (Fig.\u0026nbsp;4D; p\u0026thinsp;=\u0026thinsp;0.006). In the SVZ, we observed an increase in immature glial cells (Fig.\u0026nbsp;4E) and a trend towards an increase in OPCs (Fig.\u0026nbsp;4G; p\u0026thinsp;=\u0026thinsp;0.0524), while astrocytes remained unchanged compared to mock-infected controls at 9 dpi (Fig.\u0026nbsp;4F).\u003c/p\u003e \u003cp\u003eBecause abnormal expression of myelin proteins persisted in the surviving mice (Table\u0026nbsp;1), we next determined OPC numbers in the hippocampus and SVZ at 90 dpi. In the hippocampus, immature glial cells (A2B5\u003csup\u003e+\u003c/sup\u003e, Fig.\u0026nbsp;5A) and OPCs (O4\u003csup\u003e+\u003c/sup\u003e, Fig.\u0026nbsp;5C) were increased with infection, while astrocytes were unaltered (Fig.\u0026nbsp;5B). In the SVZ, the OPC pool increased with infection (Fig.\u0026nbsp;5F), while both the immature glia and astrocytes remained unchanged at 90 dpi (Fig.\u0026nbsp;5D and 5E, respectively). Together, these results suggest that the OPC pool undergoes long-term expansion in the hippocampus and SVZ after MeV infection in juvenile mice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eOPC proliferation remains unchanged after MeV infection\u003c/h2\u003e \u003cp\u003eAs the OPC pool increased at both 9 dpi (Fig.\u0026nbsp;4) and 90 dpi (Fig.\u0026nbsp;5), we next sought to determine the source of the new OPCs. In the postnatal brain, OPCs are one of the most proliferative cell types \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesized that OPC proliferation may account for the increased number of OPCs during infection. To quantify OPC proliferation, we determined the percentage of proliferating OPCs through bromodeoxyuridine (BrdU) labeling to mark cells actively synthesizing DNA \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. BrdU was injected daily from 6\u0026ndash;8 dpi to capture the period before 9 dpi, where we first observed an increase in OPCs (Fig.\u0026nbsp;6A). The total population of BrdU\u003csup\u003e+\u003c/sup\u003e cells and cells that co-labeled for BrdU and O4 were measured to assess all proliferating cells and proliferating OPCs, respectively (Fig.\u0026nbsp;6B). Although substantial expansion of OPC pool was detected in MeV-infected groups, both BrdU\u003csup\u003e+\u003c/sup\u003e cells and BrdU\u003csup\u003e+\u003c/sup\u003eO4\u003csup\u003e+\u003c/sup\u003e cells were unaltered with infection in the hippocampus (Fig.\u0026nbsp;6C and 6D) and the SVZ (Fig.\u0026nbsp;6E and 6F). These results indicate that the expansion in the OPC pool at 9 dpi (Fig.\u0026nbsp;4) may not be attributed to OPC proliferation \u003cem\u003eper se\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNSCs differentiate to form OPCs in both neurogenic niches during a juvenile CNS infection\u003c/h2\u003e \u003cp\u003eAnother potential source of OPCs in the brain is differentiation of NSCs in the neurogenic niches \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. We previously observed that NSC numbers transiently decline during acute infection (5 and 9 dpi), which was attributed only in part to modest cell death and differentiation into immature neurons \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Thus, we asked if the decrease in the NSC pool could also be explained by increased commitment to the OPC lineage. For these studies, we measured cells that co-labelled for OPC and NSC markers (O4 and nestin, respectively) at 5 dpi to capture events prior to the OPC expansion and myelin disturbances at 9 dpi (Fig.\u0026nbsp;7A). Using flow cytometry, we analyzed O4\u0026thinsp;+\u0026thinsp;cells within the NSC population and the total number of cells co-labeling with nestin and O4 (Fig.\u0026nbsp;7B). Within the NSC pool (nestin\u003csup\u003e+\u003c/sup\u003e cells), greater numbers of cells expressed O4 in the hippocampus by 5 dpi (Fig.\u0026nbsp;7C) and 9 dpi (Fig.\u0026nbsp;7E), suggesting that more NSCs are being driven to differentiate to OPCs. Thus, the total number of cells that express both nestin and O4 (nestin\u003csup\u003e+\u003c/sup\u003e/O4\u003csup\u003e+\u003c/sup\u003e) increased at 5 dpi in the hippocampus (Fig.\u0026nbsp;7D). However, we did not observe an increase in the total nestin\u003csup\u003e+\u003c/sup\u003e/O4\u003csup\u003e+\u003c/sup\u003e cells at 9 dpi (Fig.\u0026nbsp;7F), perhaps because the total nestin\u003csup\u003e+\u003c/sup\u003e population is reduced at this time point \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Similarly, in the SVZ, O4\u003csup\u003e+\u003c/sup\u003e cells within the NSC pool increased at 5 dpi (Fig.\u0026nbsp;7G) and thus the total number of nestin\u003csup\u003e+\u003c/sup\u003e/O4\u003csup\u003e+\u003c/sup\u003e cells also increased (Fig.\u0026nbsp;7H). At 9 dpi, we did not observe an increase in O4 expression within the NSC pool in the SVZ (Fig.\u0026nbsp;7I) and the total numbers decreased (Fig.\u0026nbsp;7J), which can be attributed to the decrease in total nestin\u003csup\u003e+\u003c/sup\u003e cells \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. We also observed that the total number of O4\u0026thinsp;+\u0026thinsp;cells does not change at 5 dpi but is increased by 9 dpi in the infected mice (Supplemental Fig.\u0026nbsp;3). Together, these results suggest that NSCs differentiate to form OPCs in both the hippocampus and SVZ as early as 5 dpi, which may eventually contribute to expansion of the OPC pool by 9 dpi.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eContinued maturation into the oligodendrocyte lineage in the brains of surviving mice\u003c/h2\u003e \u003cp\u003eSince the OPC pool increased in the surviving mice\u0026mdash;despite abnormal myelination\u0026mdash;we considered whether the OPCs failed to mature into OLs, which are ultimately responsible for myelination \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. OPCs differentiate into OLs through a multistep process by forming early OPCs, intermediate OPCs, late OPCs, and finally mature OLs \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. We utilized flow cytometry to quantify each of these stages using a series of OL markers (Table\u0026nbsp;2). For the gating strategy, Olig2, a pan oligodendrocyte marker, was used as the main gate to capture all cells in the different stages of OL differentiation (marked by a black box, Fig.\u0026nbsp;8A) \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. In both the hippocampus and the SVZ, the total number of Olig2\u0026thinsp;+\u0026thinsp;cells did not change with infection (data not shown), suggesting either that there is not an appreciable loss of OLs or that there is a drive to replace lost OLs during infection, resulting in a stable number of Olig2\u0026thinsp;+\u0026thinsp;cells.\u003c/p\u003e \u003cp\u003eWhen we examined the stages of OL differentiation in the hippocampus, we observed that early OPCs (Olig2\u003csup\u003e+\u003c/sup\u003e/PDGFRα\u003csup\u003e+\u003c/sup\u003e/A2B5\u003csup\u003e\u0026minus;\u003c/sup\u003e; Fig.\u0026nbsp;8B) and intermediate OPCs (Olig2\u003csup\u003e+\u003c/sup\u003e O4\u003csup\u003e+\u003c/sup\u003e PDGFRα\u003csup\u003e\u0026minus;\u003c/sup\u003e A2B5\u003csup\u003e\u0026minus;\u003c/sup\u003e; Fig.\u0026nbsp;8C) remained unchanged whereas late OPCs (Olig2\u003csup\u003e+\u003c/sup\u003e/O4\u003csup\u003e+\u003c/sup\u003e /Tppp-p25\u003csup\u003e+\u003c/sup\u003e /PDGFRα\u003csup\u003e\u0026minus;\u003c/sup\u003e /A2B5\u003csup\u003e\u0026minus;\u003c/sup\u003e; Fig.\u0026nbsp;8D), and OLs (Olig2\u003csup\u003e+\u003c/sup\u003e /Tppp-p25\u003csup\u003e+\u003c/sup\u003e /O4\u003csup\u003e\u0026minus;\u003c/sup\u003e /PDGFRα\u003csup\u003e\u0026minus;\u003c/sup\u003e /A2B5\u003csup\u003e\u0026minus;\u003c/sup\u003e ; Fig.\u0026nbsp;8E) increased in the surviving mice in adulthood (90 dpi). In the SVZ, we did not observe changes in the early OPCs (Fig.\u0026nbsp;8F), intermediate OPCs (Fig.\u0026nbsp;8G), and late OPCs (Fig.\u0026nbsp;8H). However, mature OLs (Fig.\u0026nbsp;8I) increased at 90 dpi in the SVZ of the surviving mice. Collectively, these data show the presence of greater OLs in both the neurogenic niches, thus showing that OPCs are propelled towards mature phenotype in surviving mice, despite the loss of myelin proteins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eNeuronal numbers remain constant despite a loss of axonal and synaptic markers after MeV infection\u003c/h2\u003e \u003cp\u003eBecause we observed disturbances in myelin proteins and OPC/OL numbers, we tested whether these disruptions were accompanied by a loss of neurons, which depend upon proper myelination for survival \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Using β-III tubulin as a marker, we quantified the number of mature neurons at 9, 30, and 90 dpi in the hippocampus by flow cytometry. Neuronal numbers were unchanged during infection (Fig.\u0026nbsp;9A-9C), despite the eventual decline in myelin markers in the hippocampus.\u003c/p\u003e \u003cp\u003eGiven that neuronal numbers were maintained, we next questioned if the maturation of neurons was compromised during infection, given that neurons are infected in our model \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. To address this question, we first investigated the expression of neuronal proteins that connect the axon to the myelin sheath, which are important for maintaining neuron-OL crosstalk and establishing the borders of the node and paranodes. Specifically, we measured contactin associated protein (Caspr), which tethers the myelin sheath at the paranode, and neurofascin-186 (NF186), which maintains the node between myelin sheaths \u003csup\u003e\u003cspan additionalcitationids=\"CR67 CR68\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. We observed that although neuronal numbers were stable, the expression of both NF186 (Fig.\u0026nbsp;9D) and Caspr (Fig.\u0026nbsp;9E) significantly decreased in the infected mice at 90 dpi. These results suggest that neuronal-OL crosstalk may be compromised after infection at 90 dpi.\u003c/p\u003e \u003cp\u003eDemyelination in the hippocampus is associated with decreased synaptic density and a loss of synaptic marker expression in postmortem MS brains \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Thus, we examined expression of the presynaptic proteins Synapsin-1 and \u0026minus;\u0026thinsp;2, which are involved in synapse formation and synaptic vesicle regulation \u003csup\u003e\u003cspan additionalcitationids=\"CR72 CR73 CR74\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. We observed a significant reduction in both synapsin-1 isoforms (1a/1b; Fig.\u0026nbsp;9F); whereas synapsin-2a trended toward a decline (Fig.\u0026nbsp;9F, p\u0026thinsp;=\u0026thinsp;0.0732) at 90 dpi. Together, our results indicate that even though neuronal numbers are stable after MeV infection, neuronal markers of appropriate myelination and synaptic function are reduced in surviving mice.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this paper, we found that juvenile mice experience enduring disruptions in myelination during a persistent MeV infection, despite a robust expansion of the OPC pool. OPCs are a highly proliferative cell type; however, we found that the expanding OPC pool arose from differentiation of neural stem cells (NSCs) into OPCs in the neurogenic niches. This sustained production of new OPCs may indicate an effort to differentiate into mature OLs to rescue myelination in the surviving hosts, as supported by increased maturation markers in OL lineage cells in infected mice. Nevertheless, surviving mice exhibit abnormal nodal and synaptic protein expression in the hippocampus, suggesting long-term damage to both neurons and myelin after acute MeV infection. Our results are consistent with findings in adult models of multiple sclerosis (MS), where OPCs are mobilized and attempt to differentiate, but remyelination is unsuccessful \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor decades, it has been speculated that viruses may act as a risk factor for demyelinating disease in susceptible hosts \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e. Recent studies have established Epstein Barr virus (EBV), a nearly ubiquitous herpesvirus, as a significant environmental risk factor in MS, with molecular mimicry between EBV proteins and myelin antigens (e.g., Glial-CAM) proposed as a mechanism for demyelination \u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e,\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Virus-induced demyelination also occurs in more acute settings (e.g. encephalomyelitis), often involving immune-mediated mechanisms, including cytolysis of infected brain cells and/or production of myelin-specific antibodies or T cells. We now appreciate that even mild peripheral infections can disturb myelination through the induction of cytokines and chemokines, such as in respiratory SARS-COV2 infection in mice \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. Although we do not yet know the immune factors that contribute to demyelination in this study, our findings suggest a slow, chronic myelin disruption in the hippocampus after MeV infection in young mice. The progressive nature of demyelination in our study bears similarities with Theiler\u0026rsquo;s murine encephalomyelitis virus (TMEV), a mouse pathogen that has been well-studied as a model of demyelinating disease. With neuroattenuated strains of TMEV, neurons are infected first during the acute infection, followed a month later by a chronic demyelinating phase with persistent infection in OLs and other glial cells \u003csup\u003e\u003cspan additionalcitationids=\"CR85\" citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. Chronic demyelination by TMEV requires the establishment of persistent infection and results in part from immunopathology against the infected OLs. We suspect that the persistent nature of MeV infection in neurons similarly creates an inflammatory environment that leads to demyelination in the CD46\u0026thinsp;+\u0026thinsp;mice. However, a key difference in our study is that MeV remains restricted to neurons in the juvenile CD46\u0026thinsp;+\u0026thinsp;mice, which may suggest a more indirect role for the immune response in our model. Our findings further show that demyelination can occur in the absence of direct OL infection by a virus in the juvenile brain.\u003c/p\u003e \u003cp\u003eMyelination and remyelination are highly dependent on neuronal activity; neurons that are electrically silenced receive poor myelination compared to electrically-active counterparts \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Thus, in our model, an alternative explanation for disrupted myelination could be that the neurons themselves are damaged, perhaps due to infection by MeV or exposure to inflammatory mediators. During acute MeV infection, juvenile CD46\u0026thinsp;+\u0026thinsp;mice express multiple cytokines (e.g., interferon-gamma (IFNγ) and TNFα) that can modulate neuronal signaling \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. IFNγ can increase inhibitory tone through release of GABA, whereas TNFα can increase neuronal excitability. In addition, MeV infection of rat cortical neurons \u003cem\u003ein vitro\u003c/em\u003e has been shown to decrease voltage-gated Ca2\u0026thinsp;+\u0026thinsp;currents \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e. One can therefore speculate that the balance of inflammatory cytokines during viral infection transiently dampens neuronal signaling and thereby deters engagement with myelinating OLs. Although we did not determine the electrical conductivity of neurons in this study, the reduction in pre-synaptic and nodal markers in surviving mice is suggestive of disturbances in neuronal signaling, which may ultimately contribute to diminished myelination or remyelination by neighboring OLs.\u003c/p\u003e \u003cp\u003eWe show that myelin disturbances in the neurogenic niches occur in conjunction with an expansion of the OPC pool, suggestive of an effort to repair myelin damage in the brain. A similar increase in NG2\u0026thinsp;+\u0026thinsp;OPCs has also been reported in an encephalogenic model of TMEV infection, where NG2\u0026thinsp;+\u0026thinsp;cell numbers and reactivity increased with infection in the hippocampi of adult mice \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e. In our model, we also found that the NSCs were contributing to the pool of O4\u0026thinsp;+\u0026thinsp;cells. This phenomenon of \u0026ldquo;fate switching\u0026rdquo; of NSCs to OPCs is seen in models of MS, other demyelinating models, and in the presence of stress hormones such as glucocorticoids \u003csup\u003e\u003cspan additionalcitationids=\"CR90\" citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u003c/sup\u003e. Although the exact molecular trigger is not yet clear, we speculate that a similar phenomenon is occurring in our model, where the stresses of the infection and inflammation induce NSC differentiation into OPCs. Intriguingly, studies in older mice suggest that OLs derived from NSCs may possess greater remyelination capacity than OLs derived directly from endogenous OPCs \u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. In a model of cuprizone-induced demyelination, NSCs in the SVZ contributed to the formation of new OLs and were essential for protection against axonal loss \u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e. Thus, the generation of OPCs from the NSC pool may represent a more effective strategy for preserving neurons during demyelination.\u003c/p\u003e \u003cp\u003eIn our study, the kinetics and extent of demyelination varied by brain region after MeV infection. Multiple variables could contribute to region-specific outcomes in the brain, including neuronal phenotypes that influence synaptic connectivity and, possibly, rates of viral uptake, the local viral load, cytokine levels, immune cell infiltration, or the stage of myelination. For instance, PLP levels increased in the cerebellum during acute infection, but resolved by 90 dpi; whereas PLP levels declined in the cortex at 9 dpi and then increased at 90 dpi. Developmental myelination generally follows a posterior to anterior pattern, where myelination is initiated and completed early in the cerebellum and relatively late in the association cortex. Thus, the stage of developmental myelination may influence the response to inflammatory stimuli or infection. In support of this possibility, influenza A infection of neonatal mice (5 days old) is similarly associated with greater MBP expression in the cerebellum at 21 dpi, which the authors interpreted as a response to hypomyelination \u003csup\u003e\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e\u003c/sup\u003e. Another explanation could be regional and temporal differences in viral load or cytokine expression after infection. Here, we demonstrated that the SVZ had a more rapid and profound loss of myelin proteins than the hippocampus. In our prior studies, the SVZ also had more MeV-infected neurons and expressed higher levels of inflammatory cytokines during acute infection than the hippocampus, which suggests that degree of demyelination may be tuned by the local level of inflammation \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA limitation of our study is that we cannot yet discern whether the loss of myelin markers is due a blockade in developmental myelination (e.g., the initial myelin sheath is not produced properly) or due to the destruction of existing myelin. Given the age of the juvenile mice at infection (10 days old), it is feasible that both mechanisms are in play. During acute infection, IFNγ is highly expressed and is required for viral control in CD46\u0026thinsp;+\u0026thinsp;mice \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e\u003c/sup\u003e. However, IFNγ has also been shown to inhibit developmental myelination by blocking OPC proliferation and maturation \u003csup\u003e\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e\u003c/sup\u003e. In the CD46\u0026thinsp;+\u0026thinsp;mice that survive to adulthood, we speculate that an autoimmune reaction may develop over time as the persistent infection is slowly resolved. Both innate immune cells (e.g., macrophage/microglia, neutrophils, and natural killer (NK) cells) and adaptive immune cells (CD4 and CD8 T cells, B cells) infiltrate the brain at 9 dpi, and B cells are still detected at 90 dpi, suggesting a sustained immune response \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Importantly, previous studies using a rodent-adapted MeV strain in rats concluded that a subacute measles encephalomyelitis (SAME) occurs after intracerebral MeV inoculation primarily due to a permissive inflammatory environment by the infection. The authors further add that this inflammatory microenvironment thus provides a conducible milieu for an autoimmune reaction to develop against myelin \u003csup\u003e\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e\u003c/sup\u003e. Thus, we speculate that myelination is disrupted at multiple levels post MeV-infection.\u003c/p\u003e \u003cp\u003eCollectively, our study highlights that CNS viral infections during early life can have long-lasting cellular and developmental consequences even when survivors reach adulthood. We show that even if the virus infects only neurons \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, the function of other brain cells can be indirectly impaired with lasting consequences for brain development. Importantly, even though demyelination was detected in multiple regions of the brain at 90 dpi, we did not note overt symptoms or neurological signs at this time point. However, over time, some of these surviving mice develop abnormal gait, partial paralysis, and seizures by 150 dpi when we found MeV to be undetectable \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, which suggests that efforts to repair or restore myelination are ultimately unsuccessful. Future studies are needed to determine the extent of behavioral and motor impairments and the quality of myelination in long-term survivors. Ultimately, our studies reveal that survival after a childhood infection does not guarantee benign cellular outcomes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003col\u003e\n \u003cli\u003eCNS: Central nervous system\u003c/li\u003e\n \u003cli\u003eADEM: Acute Disseminated Encephalomyelitis\u003c/li\u003e\n \u003cli\u003eOL: Oligodendrocyte\u003c/li\u003e\n \u003cli\u003eMBP: Myelin basic protein\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePLP: Myelin proteolipid protein\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eP10: Postnatal day 10\u003c/li\u003e\n \u003cli\u003eOPCs: Oligodendrocyte progenitor cells\u003c/li\u003e\n \u003cli\u003eNSCs: Neural stem cells\u003c/li\u003e\n \u003cli\u003eMS: Multiple sclerosis\u003c/li\u003e\n \u003cli\u003eMeV: Measles virus\u003c/li\u003e\n \u003cli\u003eSSPE: Subacute sclerosing panencephalitis\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMAG: Myelin associated glycoprotein\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eNSE: Neuron specific enolase\u003c/li\u003e\n \u003cli\u003edpi: Days post infection\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHMW: High molecular weight\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLMW: Low molecular weight\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eSVZ: Sub-ventricular zone\u003c/li\u003e\n \u003cli\u003eMOG: Myelin oligodendrocyte protein\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eFMG: FluoroMyelin Green\u003c/li\u003e\n \u003cli\u003eBrdU: Bromodeoxyuridine\u003c/li\u003e\n \u003cli\u003eCaspr: Contactin associated protein\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eEthics approval\u003c/u\u003e:\u003c/em\u003e\u0026nbsp;\u003c/strong\u003eAll animal studies were approved by the Duquesne University Institutional Animal Care and Use Committee (Protocol #1809-08 and #1865).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eConsent for publication\u003c/u\u003e:\u003c/em\u003e\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eAvailability of data and materials\u003c/u\u003e:\u003c/em\u003e\u0026nbsp;\u003c/strong\u003eThe datasets used during the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eCompeting interests\u003c/u\u003e:\u003c/em\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eFunding\u003c/u\u003e:\u003c/em\u003e\u0026nbsp;\u003c/strong\u003eThis work was supported by grants from the NIH, the State of Pennsylvania CURE Fund, and the Charles Henry Leach, III Foundation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u003cu\u003eAuthors\u0026rsquo; \u0026rsquo;contributions\u003c/u\u003e:\u003c/em\u003e\u0026nbsp;\u003c/strong\u003eYK performed the data collection, analysis, and prepared the figures. MC contributed to data collection in figures 2-6 and 8. CP, NL, and KY acquired data in figure 2 and Table 1. VS and KY assisted with mouse work in figures 2-3 and 8. AF acquired data in Figure 8. RKL contributed to statistical analyses and figures 2-3. YK and LO wrote the main manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRaper J, et al. Long-term alterations in brain and behavior after postnatal Zika virus infection in infant macaques. Nat Commun. 2020;11:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan den Pol AN, Mao G, Yang Y, Ornaghi S, Davis JN. Zika virus targeting in the developing brain. J Neurosci. 2017;37:2161\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen T, Liu G. Long-term outcome of acute central nervous system infection in children. 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Experimental measles encephalomyelitis in the rat: Generation of measles virus (MV) and T-lymphocyte cell lines specific for myelin basic protein (MBP). in Verhandlungen der Deutschen Gesellschaft f\u0026uuml;r Neurologie 472\u0026ndash;473Springer Berlin Heidelberg, Berlin, Heidelberg, (1987).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table 1 and 2","content":"\u003cp\u003eTable 1, 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"oligodendrocyte progenitor cells, oligodendrocytes, neural stem cells, measles virus, antiviral immunity, myelin, demyelination","lastPublishedDoi":"10.21203/rs.3.rs-8563506/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8563506/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eViruses can profoundly disturb myelination in the brain, leading to enduring neurological sequelae. The outcomes of neurotropic infections can be especially dire in younger children, in whom developmental myelination is underway. In some adult models of viral infection, demyelination is immune-mediated; however, it is unclear how an antiviral immune response impacts myelination in the young brain.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe investigated the outcomes of a neuron-restricted viral infection on developmental myelination in juvenile mice (10 days old), where only mature neurons are infectable by measles virus (MeV). The impact of neuronal MeV infection and the ensuing antiviral immune response on myelination was assessed during acute infection (9 days post-infection, dpi) and afterwards in surviving mice (90 dpi). We quantified myelin proteins and lipids in multiple brain regions, assessed oligodendrocyte development from the oligodendrocyte progenitor (OPC) stage to maturity, and measured neuronal markers associated with appropriate myelination and synapse formation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that (a) neuron-restricted viral infection was associated with short-term disruptions in myelin lipids and long-term disruptions in myelin proteins in multiple brain regions; (b) the OPC pool expanded in the neurogenic niches of the brain both during and after acute infection; (c) the expansion in the OPC pool originated from increased differentiation by neural stem cells (NSCs) rather than OPC proliferation; (d) oligodendrocyte maturation increased despite diminished myelination; and (e) expression of axonal and synaptic markers were compromised in surviving mice, although neuronal numbers were maintained in the hippocampus.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings show a robust OPC response in the juvenile brain during viral infection, but this response ultimately fails to normalize myelination and neuronal markers in surviving mice. We speculate that these mechanisms partly underlie life-long neurological impairments in some survivors of childhood infections.\u003c/p\u003e","manuscriptTitle":"Oligodendrocyte progenitor cells are promoted in the neurogenic niches of the juvenile mouse brain during viral infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 10:44:36","doi":"10.21203/rs.3.rs-8563506/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ca34a5f2-50aa-433f-a077-6b4e86c7be96","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-23T15:56:01+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 10:44:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8563506","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8563506","identity":"rs-8563506","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}