Oxidized phosphatidylcholines induce chronic neurodegeneration partly through IL-1β mediated positive feedback

Oxidized phosphatidylcholines induce chronic neurodegeneration partly through IL-1β mediated positive feedback | 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 Article Oxidized phosphatidylcholines induce chronic neurodegeneration partly through IL-1β mediated positive feedback Ruoqi Yu, Brian Lozinski, Ally Seifert, Khanh Ta, Stephanie Zandee, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4792293/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Nature Neuroscience → Version 1 posted You are reading this latest preprint version Abstract Oxidized phosphatidylcholines (OxPC) are neurotoxic byproducts of oxidative stress elevated in the central nervous system (CNS) during progressive multiple sclerosis (P-MS). How OxPC contribute to the pathophysiology of P-MS is unclear. Here, we report that OxPC deposition in the CNS of mice induces a chronic compartmentalized lesion with pathological features similar to chronic active lesions found in P-MS. Using this new model, we found that while microglia protected the CNS from chronic neurodegeneration, they were also replaced by monocyte derived macrophages in chronic OxPC lesions. Aging, a risk factor for P-MS, altered microglial composition and exacerbated neurodegeneration in chronic OxPC lesions. Amelioration of disease pathology in caspase 1/4 deficient mice and by blockade of IL-1R1 indicate IL-1β signaling contributes to chronic OxPC accumulation and neurodegeneration. These results highlight OxPC and IL-1β as potential drivers of chronic neurodegeneration in MS and suggest that their neutralization may be effective for treating P-MS. Biological sciences/Neuroscience/Neuroimmunology Biological sciences/Neuroscience/Diseases of the nervous system/Multiple sclerosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Chronic neurodegeneration defined as the continuous degradation of neuronal structure and function, as well as neuronal death, is significantly associated with disability accumulation and mortality in people living with progressive multiple sclerosis (P-MS) 1 – 5 . Compared to relapsed-remitting MS in which disease activity is largely driven by peripheral immune cells that infiltrate the CNS 1 , 6 , 7 , formation of new lesions associated with blood-brain barrier damage is less frequent in P-MS. Instead, there is an increased number of mixed active-inactive/chronic active lesions (CAL) 8 , 9 , which are characterized by a relatively inactive lesion core surrounded by a radially expanding rim with reactive microglia/macrophages, demyelination, and neuroaxonal loss 1 , 9 . MRI and post-mortem analysis indicate CAL are sites of chronic neurodegeneration that enlarge over months to years, and they are associated with worsening disability and MS progression 3 , 10 – 15 . However, because the molecular mechanisms that promote chronic neurodegeneration and CAL expansion are relatively unknown, developing effective treatments against disease progression and disability accumulation in P-MS remains challenging 2 – 4 , 16 . The elevation of oxidative stress byproducts such as oxidized phosphatidylcholines (OxPC) with degenerating neuronal cells and axons in P-MS 17 – 22 and the accumulation of reactive microglia/macrophages with phenotypes associated with inflammation and free radical production at the rim of expanding CAL 8 , 9 , 14 , 15 , 18 , 23 – 25 suggest that oxidative injury contributes to chronic neurodegeneration in P-MS. We previously demonstrated OxPC found in relapse-remitting MS lesions mediate acute neuroinflammation and neurodegeneration in the spinal cord white matter (SCWM) of young and middle-aged mice 26 – 28 , suggesting OxPC may be key mediators of tissue injury in MS. Yet, how OxPC affect chronic neurodegeneration in P-MS is not known because commonly used rodent models of MS express relatively low levels of OxPC compared to human MS 17 – 19 , 26 . Given OxPC are implicated as key mediators of tissue degeneration in chronic non-CNS diseases 29 – 32 and that they accumulate in brain lesions throughout different stages of MS progression 17 , 20 , 21 , 27 and even in deep GM where neurodegeneration commonly occurs 22 , it is imperative to investigate the role of OxPC in P-MS. Here, we aimed to determine the potential of acute OxPC injuries progressing to mediate chronic neurodegeneration in the mouse SCWM. Our results highlight that OxPC deposition uniquely promotes a chronic lesion with compartmentalized neuroinflammatory and neurodegenerative features similar to CAL in P-MS. Notably, we found aging, microglia, and IL-1β/IL-1 receptor type I (IL-1R1) signaling are key factors that modulate the intensity of OxPC mediated chronic neurodegeneration. RESULTS OxPC induce chronic neuroinflammation, demyelination, and neurodegeneration Previous imaging mass spectrometry experiments indicate that 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) is one of the most abundant species of OxPC in active MS lesions and that its deposition in the CNS mediates acute neuroinflammation, demyelination, and neurodegeneration 26 . To determine if acute focal lesions induced by POVPC progress to become sites of chronic neurodegeneration akin to CAL in P-MS, we first induced lesions in the SCWM of 6-week-old mice by a one-time stereotactic injection of POVPC and compared tissue pathology after 7 and 42 days (Fig. 1 A). Unlike the experimental autoimmune encephalomyelitis (EAE) model of MS neuroinflammation where lesions form stochastically and are therefore difficult to assess over time 33 , and the lysolecithin (LPC) model of MS focal demyelination where remyelination and repair occurs relatively quickly 34 – 36 , the focal lesion induced by POVPC remained chronically inflammatory and demyelinated after 42 days (Fig. 1 B-D). While day 42 chronic lesions were smaller in area compared to day 7 acute lesions (Fig. 1 E), scar forming glial fibrillary acidic protein (GFAP) + astrocytes at the lesion border and the intralesional accumulation of CD11b + mononuclear phagocytes were similar between chronic and acute lesions (Fig. 1 C, F). The lack of myelin basic protein (MBP) in day 42 lesions also indicates remyelination failure despite an increase in oligodendrocyte transcription factor (OLIG2) + oligodendroglia 37 (Fig. 1 B, I, J). Interestingly, the percent area of lesional IBA1 + cells and the average size of these cells were reduced at day 42 compared to day 7 (Fig. 1 B, G, H), suggesting that microglia/macrophage composition or behavior is different in chronic lesions compared to acute lesions. Indeed, the levels of lesional P22phox subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and interleukin (IL)-β, as well as the proportion of CD11b + that overlapped with these two inflammatory molecules, were elevated at day 42 compared to day 7 (Fig. 1 C, D, K-N). OxPC can be specifically bound and detected by the natural IgM antibody E06 29, 38 . Since we previously found minimal E06 + immunoreactivity in acute lesions at 24 h post POVPC injection 26 , the elevation of E06 + immunoreactivity thereafter represents the accumulation of endogenously produced OxPC. Thus, similar levels of E06 + immunoreactivity in day 7 and day 42 lesions suggested that the accumulation of endogenous OxPC was sustained in chronic lesions (Fig. 2 A, E). To assess neurodegeneration in chronic OxPC lesions, we compared neurofilament heavy chain (NFH) + axons between day 7 and day 42 (Fig. 2 A, B). Unlike NFH + axons in the normal appearing white matter (NAWM), axons within day 7 lesions appear enlarged and were more dispersed across the lesion, which suggested edema and axonal stress. In contrast, NFH + axons are mostly absent in the core of day 42 lesions, which suggests neurodegeneration had occurred; axons that remain are largely found near the lesion edges and are similar in size as axons in the NAWM. The significant decrease of lesional NFH + area relative to NAWM NFH + area and average NFH + axon size in day 42 lesions compared to day 7 lesions supports these observations (Fig. 2 F, G). Furthermore, the proportion of NFH + axons that overlapped with beta amyloid precursor protein (βAPP), a marker of axonal injury 39 , trended to be higher in chronic lesions compared to acute lesions (Fig. 2 H). Additionally, while βAPP + NFH + axons were found mostly at lesion cores at day 7, they were found more at the lesion edges by day 42 (Fig. 2 B, I), which suggests an outward expansion of axonal injury from the lesion core. Interestingly, compared to acute lesions, chronic lesions also had significantly less CD11b + Ki67 + proliferative cells (Fig. 2 C, J, K) but significantly more IBA1 + cells that were labeled with phosphorylated histone (pH2A.X) (Fig. 2 D, L, M), which is a marker of DNA damage and repair 40 . These observations are additional evidence that mononuclear phagocytes found in chronic lesions were in distinct cellular states compared to those found in acute lesions. Collectively, these results indicated that the deposition of MS-relevant OxPC, such as POVPC, in the CNS induced a CAL-like focal lesion with endogenous OxPC accumulation as well as compartmentalized chronic neuroinflammation, demyelination, and neurodegeneration. Notably, OxPC induced chronic lesions remained inflamed and unrepaired after 100 days (Extended Fig. 1 A-E). OxPC induced chronic neurodegeneration features endogenous lipid peroxidation and reemergence of monocyte derived macrophages (monoMac) Reactive microglia/macrophages found in CAL associate with disease activity and lesion expansion in P-MS 9 , 41 . However, the differential contribution of microglia versus monoMac to CAL disease activity and MS progression remains difficult to study. To address this knowledge gap, we next utilized the chronic OxPC lesions to interrogate the ontogeny of the mononuclear phagocytes during chronic neurodegeneration using CX3CR1 CreER :Ai9 tdTom mice. These mice were first treated with tamoxifen 3 weeks before the experiment to label long-lived microglia with the tdTomato fluorescent reporter protein and to allow for the short-lived tdTomato labeled monocytes to turnover. POVPC was then injected into the mouse SCWM and the proportion of microglia (IBA1 + tdTomato + ) versus monoMac (IBA1 + tdTomato − ) was analyzed in chronic lesions at day 42. Additionally, separate groups of CX3CR1 CreER :Ai9 tdTom mice were injected with an equivalent amount of LPC to determine how chronic OxPC lesions differ from the LPC induced focal demyelination (Fig. 3 A). By day 42, the overall size of the chronically demyelinated lesion area marked by MBP disruption (Fig. 3 A-E, K) and the amount of total lesional IBA1 + microglia/macrophage (Fig. 3 F) were similar between OxPC and LPC injected mice. Unlike day 7 acute OxPC 27 and LPC lesions 42 where > 90% of lesional macrophages are tdTomato + microglia, only approximately 50% of IBA1 + cells from chronic OxPC and LPC lesions overlapped with tdTomato (Fig. 3 H), which suggested an increase contribution of monoMac to the total macrophage population within chronic focal lesions in the CNS. Interestingly, IBA + tdTomato + microglia in OxPC lesion were significantly larger in size compared to microglia in LPC lesions (Fig. 3 I, J), suggesting that microglia in chronic OxPC lesions may be more representative of enlarged/amoeboid reactive microglia found in CAL from P-MS 43 , 44 . More importantly, significant endogenous OxPC deposition was only found in chronic OxPC lesions but not in chronic LPC lesions (Fig. 3 B, G) and OxPC was only minimally detected near the meninges of spinal cord and cerebellum lesions from day 45 chronic EAE mice (Extended Fig. 2 A, B). Furthermore, the remyelinating potential and chronic neurodegeneration marked by OLIG2 + oligodendroglia accumulation and NFH + axon loss, respectively, were significantly worse in chronic OxPC lesions compared to chronic LPC lesions (Fig. 3 L, M). Thus, endogenous lipid peroxidation, chronic neurodegeneration, and the equal accumulation of reactive microglia and monoMac are unique pathological features of chronic OxPC lesions that may be informative for CAL expansion and P-MS. Aging exacerbates OxPC induced chronic neurodegeneration Aging is a major risk factor for neurodegenerative diseases 45 and P-MS onset most often occurs during middle age 5 . To investigate how aging changes the susceptibility of the CNS to OxPC mediated chronic neurodegeneration, we induced OxPC deposition in the SCWM 6wk old young mice and 52wk old middle-aged mice and analyzed chronic neurodegeneration (Fig. 4 A). Compared to 6wk old mice, day 42 chronic lesions in 52wk old mice had greater lesion spread and significantly greater total lesion volume (Fig. 4 B-E). While there was a significant reduction in lesion spread at day 42 compared to day 7 in 6wk old mice, this did not occur in 52wk old mice (Fig. 4 F), suggesting that the response to limit OxPC mediated chronic neurodegeneration became less effective in the aging CNS. Indeed, there was significantly greater accumulation of endogenous E06 + OxPC as well as OxPC that overlapped with IBA1 + microglia/macrophages in 52wk old chronic lesions compared to 6wk old chronic lesions (Fig. 4 G, J-L). Moreover, while there were no differences in IBA1 + microglia/macrophages, OLIG2 + oligodendroglia, and overall amount of MBP between chronic lesions from 6wk old and 52wk old mice (Fig. 4 H, M, N), the density of surviving NFH + axons in lesions from middle-aged mice were significantly lower than axons in lesions from young mice (Fig. 4 H, O). In addition, the average size of NFH + axons, the level of βAPP, and the proportion of NFH + axons that overlap with βAPP + in chronic lesions from 52wk old mice were significantly greater compared to chronic lesions from 6wk old mice (Fig. 4 I, P-R). These observations indicate that aging exacerbates chronic neurodegeneration and endogenous OxPC accumulation in the CNS. Aging promotes microglial dysfunction during OxPC induced chronic neurodegeneration We previously found that compared to 6wk old mice, microglia/macrophages from 52wk old mice had significant transcriptional and functional reprogramming in response to acute OxPC mediated neurodegeneration 27 , 28 . To investigate if aging also causes microglial dysfunction during chronic OxPC mediated neurodegeneration, we further profiled the functional phenotype of microglia/macrophages found in day 42 lesions of 6wk old and 52wk old mice. While there was no difference in total IBA1 + cells between young and middle-aged lesions (Fig. 4 K), there was greater accumulation of CD11b + cells in middle-aged chronic lesions compared to young lesions (Fig. 5 A, C), suggesting that aging may alter microglia/macrophage composition in chronic lesions. In addition, P22phox and IL-1β, which were upregulated by microglia/macrophages in day 42 chronic lesions compared to day 7 acute lesions (Fig. 1 K-N), were also significantly elevated in middle-aged lesions compared to young lesions (Fig. 5 D-G). Middle-aged lesions also had significantly greater amount of p16 INK 4 a (Extended Fig. 3 A, D), a marker of senescence in the aging CNS 46 . In contrast, the expression of induced nitric oxide synthase (iNOS) and arginase 1 (Arg1) were relatively low in chronic lesions and there were no differences between young and middle-aged mice (Extended Fig. 3 B, C, E-H). These observations suggest that aging promoted inflammatory dysregulation in microglia/macrophages during chronic neurodegeneration. The composition of macrophages in day 42 chronic OxPC lesions was approximately 50% microglia and 50% monoMac (Fig. 3 H) whereas macrophages in day 7 acute lesions were > 90% microglia 27 . Since increased engraftment of monoMac in the CNS associates with aging and neurodegenerative diseases 47 , we next analyzed day 42 chronic OxPC lesions in tamoxifen treated 6wk old and 52wk old CX3CR1 CreER :Ai9 tdTom mice (Fig. 5 H) to investigate whether aging promotes greater replacement of microglia by monoMac in chronic OxPC lesions. Like 6wk old and 52wk old wildtype mice, there was no difference in the amount of IBA1 + cells between 6wk old and 52wk old CX3CR1 CreER :Ai9 tdTom mice (Fig. 5 I, J). However, the abundance of IBA1 + tdTomato + microglia were significantly reduced in 52wk old CX3CR1 CreER :Ai9 tdTom mice compared to 6wk old mice (Fig. 5 L, M). Furthermore, despite increased OxPC accumulation in chronic lesions of 52wk old CX3CR1 CreER :Ai9 tdTom mice compared to 6wk old mice (Fig. 5 K), the proportion of E06 + OxPC that overlapped with tdTomato + microglia was significantly lower in middle-aged lesions compared to young lesions (Fig. 5 N). Together, these results suggest aging facilitated greater monoMac accumulation and reduced microglial sequestration of OxPC in chronic OxPC lesions. Microglia depletion in chronic OxPC lesions worsens neurodegeneration Although post-mortem analysis of CAL found in P-MS brains highlight the involvement of reactive, proinflammatory microglia/macrophages 9, 41 , the pathological role of microglia within CAL and P-MS remains less well understood. Since OxPC deposition in the CNS induces a CAL-like focal lesion with both microglia and monoMac, we next utilized this model to study the functional significance of microglia during chronic oxidative damage, neuroinflammation, and neurodegeneration. Specifically, CX3CR1 CreER :Rosa26 iDTR mice were treated with tamoxifen to induce diphtheria toxin receptor (DTR) expression in long-lived microglia and to allow short-lived DTR + monocytes to turnover. Three weeks later, the SCWM of these mice were then injected with POVPC to induce the focal OxPC lesion, followed by PBS or diphtheria toxin (DT) treatment once every other day between days 0–7 or 35–42 after OxPC deposition (Fig. 6 A). We aimed to compare how DT mediated microglia depletion during the acute phase of the lesion (days 0–7) or during the chronic phase of the lesion (days 35–42) differentially impacted the outcome of chronic neurodegeneration at day 42. Unexpectedly, mice with DT treatment during acute neurodegeneration (days 0–7) failed to survive past day 18 (Fig. 6 B), perhaps due to worsening of acute neurodegeneration 48 or non-specific long-term toxicity from DT 49 . Thus, how the acute microglial response to OxPC deposition regulated the pathology of the chronic lesion could not be determined. For mice with DT treatment during chronic neurodegeneration (days 35–42), there was no difference in overall lesion volume by day 42 compared to mice treated with PBS (Fig. 6 C). However, within the lesions, DT treated mice had significantly reduced amount of total IBA1 + cells compared to PBS treated mice (Fig. 6 D, H), indicative of microglial depletion. While there was no difference between PBS and DT treated mice in the amount of endogenous E06 + OxPC deposition (Fig. 6 I), the overlap of E06 + OxPC with IBA1 + cells was significantly less in lesions from DT treated mice compared to lesions from PBS treated mice (Fig. 6 J), suggesting reduced OxPC clearance by microglia. Interestingly, the overall lesional accumulation of CD11b + cells within PBS and DT treated mice was not significantly different (Fig. 6 E, K), suggesting that monoMacs may have replaced microglia in DT treated mice. Moreover, while the level of lesional P22phox and the proportion of CD11b + cells that overlapped with P22phox were similar between PBS treated and DT treated mice, the level of IL-1β and the proportion of IBA1 + cells that overlapped with IL-1β were significantly elevated in DT treated mice compared to PBS treated mice (Fig. 6 F, L-O). Furthermore, DT treatment significantly reduced the amount of lesional MBP as well as the density of NFH + axons and OLIG2 + oligodendroglia (Fig. 6 G, P-R) in lesions compared to PBS treatment. Collectively, these results showed that the depletion of microglia in chronic OxPC lesions abnormally elevated IL-1β, impaired remyelination potential, and exacerbated neurodegeneration. Progressive MS and severity of chronic neurodegeneration associate with OxPC accumulation and IL-1β activity Thus far, we demonstrated that POVPC deposition in the SCWM of mice induces a CAL-like unremitting lesion with endogenous OxPC generation as well as chronic neuroinflammation and neurodegeneration, but the endogenous mechanism that drive this chronic pathology remain unclear. Interestingly, OxPC can promote IL-1β secretion by myeloid cells including macrophages 29 , 50 , 51 and we previously found IL-1β deposition in the SCWM of mice induced acute endogenous OxPC formation 26 . Since total IL-1β (Fig. 1 M) and cleaved IL-1β (Extended Fig. 4 A-D) were elevated in chronic OxPC lesions compared to acute lesions, and that IL-1β was further dysregulated when OxPC mediated chronic neurodegeneration was exacerbated by aging (Fig. 5 F) or by microglia depletion (Fig. 6 N, O), we hypothesized that OxPC deposition may promote a vicious cycle of chronic neuroinflammation and oxidative stress at least in part through aberrant IL-1β production and/or signaling. Since caspase 1/4 (CAS1/4) deficiency prevents the processing of pro-IL-β to its active form 52 , 53 , we tested this hypothesis by inducing chronic OxPC lesions in CAS1/4 +/+ and CAS1/4 −/− mice. Although the size of the lesion epicenters at day 42 were similar between CAS1/4 +/+ and CAS1/4 −/− mice, the total volume of chronic lesions from CAS1/4 −/− mice trended to be smaller compared to chronic lesions from CAS1/4 +/+ mice (Fig. 7 A, D, E). Although there was no difference in the amount of CD45 + cells, CAS1/4 −/− mice had significantly lower amounts of cleaved IL-1β and percent of CD45 + cells that overlapped with cleaved IL-1β compared to CAS1/4 +/+ (Fig. 7 B-G), which is consistent with their deficiency in CAS1/4 expression. In addition, while the amount of lesional IBA1 and MBP were not different between the two groups, chronic lesions from CAS1/4 −/− mice had significantly reduced amount of E06 + OxPC accumulation and loss of NFH + axons compared to chronic lesions from CAS1/4 +/+ mice (Fig. 7 C, D, H-M). These results suggest that CAS1/4 activity contributes to endogenous lipid peroxidation and chronic neurodegeneration. Since IL-1β signaling is dependent on IL-1R1 54 and can promote neuroinflammation 55 , we also treated mice with PBS or anakinra, an antagonist to IL-1R1 from days 28–42 following OxPC deposition in the SCWM (Fig. 8 A). Compared to chronic OxPC lesions in PBS treated mice, chronic lesions in anakinra treated mice had significantly reduced amount of E06 + OxPC accumulation (Fig. 8 B, D) but significantly more IBA1 + microglia/macrophages, which overlapped with E06 + OxPC (Fig. 8 E, F). While lesional MBP percentage was equivalent in both treatment groups (Fig. 8 C, G), anakinra treated chronic lesions had significantly greater density of OLIG2 + oligodendroglia (Fig. 8 H) and NFH + axons (Fig. 8 I) compared to PBS treated lesions. Thus, blockade of IL-1R1 in chronic OxPC lesions reduced OxPC accumulation and partially ameliorated the severity of chronic neurodegeneration. Finally, to validate the relevancy of OxPC deposition and IL-1β elevation in P-MS, we analyzed CD45 and E06 immunoreactivity in the NAWM and the CAL rims from fresh-frozen tissues collected from rapid autopsies of P-MS brains (Fig. 8 J). Compared to CD45 + cells that resembled more ramified microglia in the NAWM, CD45 + cells in the rims of CAL were significantly larger and more amoeboid in morphology (Fig. 8 K-M). Importantly, CAL rims had significantly greater deposition of E06 + OxPC as well as significantly greater amount of E06 + OxPC that overlapped with CD45 + cells (Fig. 8 N, O). Total IL-1β and IL-1β + CD45 + cells were also elevated in CAL rims compared to the NAWM (Fig. 8 P, Q). Thus, endogenous OxPC deposition as well as reactivity of IL-1β and OxPC associated microglia/macrophages were pathological features of CAL in P-MS. DISCUSSION The molecular mechanisms mediating chronic neurodegeneration and MS progression are not well understood. OxPC are neurotoxic byproducts of oxidative stress and OxPC accumulation prominently associates with all stages of MS 17 , 20 , 21 , 26 . Here, we report that OxPC deposition in the mouse SCWM induced a chronic focal lesion with features akin to CAL from P-MS including compartmentalized neuroinflammation, demyelination, and neurodegeneration, as well as endogenous OxPC accumulation. The severity of chronic neurodegeneration and endogenous OxPC accumulation in these CAL-like lesions were significantly increased by aging and by microglia depletion. An increase of IL-1β levels was associated with lesion chronicity and with conditions that exacerbated chronic neurodegeneration and endogenous OxPC accumulation, suggesting a link between OxPC mediated pathology and IL-1β activity. Indeed, E06 + OxPC immunoreactivity as well as the loss of NFH + axons were significantly lower in CAS1/4 −/− mice, which have deficiency in pro-IL-1β processing 52 , 53 . Conversely, IL-1R1 blockade ameliorated axon loss and endogenous OxPC accumulation in chronic lesions. These results together with the observation that IL-1β and OxPC accumulated in CAL from P-MS brains, suggested that aberrant IL-1β signaling and OxPC contributes to chronic neurodegeneration in P-MS. Although OxPC accumulation 17 – 22 and inflammation associated oxidative injury 8 , 9 , 14 , 15 , 18 , 23 – 25 are major features of MS pathophysiology, their functional involvement in P-MS is not well understood because commonly studied experimental models of MS such as EAE or LPC induced demyelination have pathologies that involve relatively low levels of OxPC and oxidative injury 18 , 19 . Here, we show that a one-time deposition of POVPC, a type of OxPC upregulated in MS lesions 26 , promoted chronic unremitting compartmentalized injury in the SCWM of mice. Chronic OxPC lesions had significant neurodegeneration in the form of axonal loss, persistent demyelination/remyelination failure, as well as accumulation of endogenous OxPC and inflammatory microglia/monoMac that upregulated NADPH oxidase and IL-1β. These pathological responses are similar to CAL disease activity in P-MS, which also involves axon loss 56 , ongoing demyelination 57 , microglia/monoMac reactivity 9 , and NADPH oxidase upregulation 25 . Moreover, we found significant accumulation of OxPC and IL-1β with reactive phagocytes in CAL from P-MS brains. These observations indicate that OxPC mediated chronic lesions recapitulated key aspects of P-MS pathophysiology. Notably, the exacerbation of OxPC mediated chronic neurodegeneration in middle-aged mice is evidence that aging associated defects in OxPC mitigation contributes to the acceleration of chronic neurodegeneration as people with P-MS become older. Thus, this model will be useful for uncovering new mechanisms that regulate chronic neurodegeneration in P-MS and aging. The concentration of OxPC deposited in active lesions or CAL from MS have not been measured systematically and only one study estimated there are approximately 0.6 µg of OxPC per mg of protein isolated from MS brain homogenate 58 . In this study, chronic lesions developed after the deposition of 5 µg of POVPC, which is likely within the range of OxPC concentrations potentially found in MS lesion microenvironments. Using the OxPC specific and neutralizing E06 antibody 29 , 30 , 38 , we previously found minimal OxPC in the focal SCWM lesion at 1 day post POVPC injection 26 , which indicates that most if not all of the injected POVPC quickly reacts with the tissue. Thus, the detection of E06 + OxPC within focal OxPC lesions after 42 and 100 days reflects significant endogenous OxPC production, potentially due to similar mechanisms found in MS lesions. Notably, OxPC accumulation was minimal in chronic LPC or EAE lesions, highlighting that the pathology in these models may be different from that of chronic OxPC lesions and OxPC + CAL from P-MS. The cause of the endogenous OxPC accumulation in MS lesions remains relatively unknown. Reactive microglia/monoMac are major populations of immune cells found in CAL of P-MS and they express NADPH oxidase 17 – 19 , 25 . Thus, they have the capacity to produce ROS, which can promote nonenzymatic oxidation of PC into OxPC 30 in the lesion microenvironment. Similarly, microglia and monoMac with elevated NADPH oxidase expression were also the predominant immune cells in chronic OxPC lesions, suggesting they may contribute to lesion chronicity by producing ROS which leads to lipid peroxidation and endogenous OxPC formation, which in turn generate a feed-forward loop to generate more ROS and OxPC 30 . Alternatively, NADPH oxidase upregulation by microglia/monoMac in chronic lesions may not fully reflect in situ ROS/OxPC generation as PC oxidation can also be initiated enzymatically by 12/15-lipoxygenase 59, 60 , which is also upregulated in MS 61 . Both of these possibilities may be investigated by generating transgenic mice with microglia/monoMac deficient in NADPH oxidase 62 or 12/15-lipoxygenase 63 expression. Nevertheless, since IL-1β was significantly elevated in chronic OxPC lesions and in CAL from P-MS, it may be involved in sustaining microglia/monoMac reactivity, oxidative stress, and lipid peroxidation in the lesion microenvironment. In support of this hypothesis, we previously showed IL-1β deposition in the SCWM directly promotes acute neuroinflammation and endogenous OxPC formation 26 whereas here we found blocking IL-1R1 signaling reduced OxPC mediated chronic neurodegeneration. These findings, together with recent evidence showing that OxPC can also enhance IL-1β production in both primed myeloid cells 50 , 51 , 64 and mouse peritoneal macrophages 29 suggest a potential positive feedback loop between OxPC and IL-1R1 mediated inflammatory responses. Thus, strategies to inhibit IL-1β signaling in P-MS may help to interrupt the vicious cycle of OxPC generation, neuroinflammation, and neurodegeneration. It is important to note that OxPL are abundantly generated whenever cells undergo apoptosis cell death, thus further contributing to this positive feed-forward destructive cycle 65 , 66 . CAL associate with chronic neurodegeneration and disability progression in people with P-MS 8 , 57 , 67 . They are classified by a relatively inactive lesion core surrounded by a slowly-expanding lesion rim with ongoing myelin loss and accumulation of lipid-laden microglia/monoMac 1, 41 , 67 . The relative composition of microglia versus monoMac in CAL remains unclear and is difficult to determine as the expression of microglia specific markers such as TMEM119 or P2RY12 becomes less stable in reactive microglia 68 . While microglia and monoMac in CAL are largely characterized as proinflammatory cells 9 , 15 , 68 , their functional contribution to chronic neurodegeneration remains uncertain. By fate mapping tdTomato + microglia in chronic OxPC lesions, we found that unlike in acute lesions 26 , 27 , 42 , tdTomato − monoMac re-emerged as a significant immune cell population in chronic OxPC lesion. Importantly, the amount of monoMac versus microglia was significantly increased within chronic lesions of middle-aged mice compared to lesions from young mice, which is consistent with recent findings showing that chronic injury and aging drive the accumulation of disease associated monoMac in the CNS 47 , 69 . Importantly, we found that middle-aged mice with greater monoMac accumulation and mice with microglia depletion had greater OxPC and IL-1β accumulation as well as worse chronic neurodegeneration compared to young mice and mice without microglia depletion, respectively. These findings together with studies showing that microglia mediated phagocytic processing of harmful lipids becomes dysregulated by aging 26 , 27 , 70 – 73 , implicate that microglial loss and/or their dysregulation caused by chronic disease activity and aging can facilitate the acceleration of chronic neurodegeneration in P-MS. In summary, we report that OxPC deposition in the CNS induces pathology similar to CAL from P-MS including endogenous lipid peroxidation, unremitting neuroinflammation, and chronic neurodegeneration. Aging and microglial dysregulation in part through inflammatory IL-1β signaling exacerbated chronic neurodegeneration. Promoting OxPC neutralization and clearance, as for example by targeting with the E06 antibody, and/or promoting homeostatic microglia repopulation may be effective approaches to slow and/or halt chronic neurodegeneration in P-MS. ONLINE METHODS MS specimens Post-mortem human brain tissues were obtained from seven patients diagnosed with clinical and neuropathological P-MS according to the revised 2010 McDonald’s criteria 67 , 74 , with full ethical approval (BH07.001, Nagano 20.332 - YP) and informed consent as approved by the CRCHUM and University of Montreal research ethics committee. As previously described 75 , 76 , autopsy samples were cryopreserved, and lesions were classified using Luxol fast blue and H&E staining. Mice All experiments were conducted with ethics approval (protocol number 20220103) from the Animal Care Committee at the University of Saskatchewan under regulations of the Canadian Council of Animal Care. Female 6wk and 52wk old C57Bl/6J mice were acquired from Jackson Laboratories for in vivo experiments. CX3CR1 creER (strain 021160) mice, Ai9 TdTom mice (strain 007909), and Rosa26 iDTR mice (strain 007900) from The Jackson Laboratory were bred in the Lab Animal Services Unit at the University of Saskatchewan to produce male and female CX3CR1 CreER :Ai9 tdTom mice and CX3CR1 CreER :Rosa26 iDTR mice for microglial fate mapping and depletion studies. Female 6-10wk old C57BL/6NJ (CAS1/4 +/+ , strain 005304) and B6N.129S2-Casp1tm1Flv/J (CAS1/4 /− strain 016621) mice from Jackson Laboratories were used for IL-1β studies. Mice were maintained on a regular diet in low humidity environment on a 12-hr light/dark cycle at 21 to 23 degrees Celsius with unlimited access to food and water. Mice and littermates were randomly assigned to different experimental groups. Fate mapping microglia/monoMac Six-week-old and 52-week-old CX3CR1 CreER :Ai9 tdTom mice were intraperitoneally injected with 2 mg of tamoxifen (20 mg/ml; T5648, Sigma) dissolved in corn oil (C8267) once a day for 3 consecutive days to induce tdTomato expression in all CX3CR1 + mononuclear phagocytes. Mice were then used 4 weeks after tamoxifen injection which is when tdTomato expression only labels long lived CNS resident microglia and macrophages but not monoMac derived from infiltrating monocytes. Microglia depletion Newly weaned CX3CR1 CreER :Rosa26 iDTR mice were injected intraperitoneally with 2 mg tamoxifen (20 mg/ml; T5648, Sigma) dissolved in corn oil (C8267) once a day for 5 consecutive days to induce DTR expression on microglia and were used for experiments 3 weeks after tamoxifen injection. For microglia depletion during acute lesion phase, tamoxifen treated CX3CR1 CreER :Rosa26 iDTR mice were injected intraperitoneally with PBS or 1 µg of DT every other day from days 0 to 7 after OxPC deposition in the SCWM. For microglia depletion during chronic lesion phase, tamoxifen treated CX3CR1 CreER :Rosa26 iDTR mice were injected intraperitoneally with PBS or 1 µg of DT every other day from days 35 to 42 after OxPC deposition. Anakinra blockade of IL-1R1 Six-week-old C57Bl/6J mice were intraperitoneally injected daily with 100 µl of PBS or PBS containing 1 mg/kg anakinra (HY-108841, MedChemExpress) from days 28–42 following OxPC deposition in the SCWM. Spinal cord surgery The surgical procedure for OxPC spinal cord injection was performed as described from previous studies 26 , 27 , 77 . Briefly, mice were anesthetized with ketamine and xylazine and injected stereotactically with 0.5 µl PBS containing 10 mg/ml POVPC (Avanti Polar Lipids, 870606P) into the ventrolateral SCWM between the T3 and T4 vertebra. After the injection, the needle was left in place for 2 min to prevent back flow, and then the mouse was sutured and placed in a thermally controlled environment for recovery. Alternatively, equivalent amount of LPC (Sigma, L1381) was injected in the SCMW using the same approach. EAE and tissue isolation Eight- to 10-week-old female C57Bl/6J mice were subcutaneously inoculated with 50 µg of MOG 35–55 peptide (Protein and Nucleic Acid Facility, Stanford University School of Medicine) in 100 µl of complete Freund’s adjuvant supplemented with 4 mg/ml heat-inactivated Mycobacterium tuberculosis H37Ra (Sigma-Aldrich), in which 50 µl emulsion was deposited on each side of the tail base. Intraperitoneal injection of pertussis toxin (300 ng per 200 µl; 180, List Biological Laboratories) was performed days 0 and 2 after MOG immunization. Mice were monitored and scored daily on a scale of 0–15. EAE mice during chronic disease (day 45) were euthanized with intraperitoneal injections of ketamine and xylazine. Fifteen ml of PBS was then perfused via cardiac puncture, and the cerebellum and spinal cord were collected. The cerebellum was frozen in Optimal cutting temperature polymer (Leica), whereas the spinal cord was processed as stated above for the spinal cord injections. Cerebellar sections (sagittal) and the spinal cord sections (longitudinal) were cut into 20-µm sections using a cryostat and collected onto Superfrost Plus microscope slides (VWR) and stored at − 20°C before analysis. Spinal cord tissue isolation for histology and microscopy analysis Mice were euthanized with intraperitoneal ketamine and xylazine overdose after 7-, 42-, or 100-days post-surgery. Ten ml of PBS followed by 10 ml of 4% paraformaldehyde in PBS were perfused via cardiac puncture. The spinal cord was then dissected from the back of the mouse, and the tissue containing the T3-T4 inject site was collected into 4% paraformaldehyde in PBS for fixation overnight at 4 degrees. Thereafter, spinal cords were transferred to 30% sucrose solution for dehydration for at least 48h and frozen in FSC 22 Frozen Section Media (Leica). With a cryostat (ThermoFisher Scientific), spinal cord tissue was cut into 20 µm coronal sections and collected on to Superfrost Plus microscope slides (VWR). Tissues were stored at -20 degrees prior to staining and analysis. Antibodies The following primary antibodies were used for immunofluorescence microscopy: mouse IgM E06 anti-OxPC (5 µg/ml, generously provided by Witztum and Tsimikas labs), rabbit anti-human/mouse IBA1 (1:1000, Wako 019-19741), chicken anti-human/mouse IBA1 (1:1000, Synaptic Systems 234 009), rat anti-mouse MBP (1:200, Abcam ab7349), rat anti-mouse CD11b (1:200, ThermoFisther 14-0112-82), goat anti-human/mouse OLIG2 (1:200, R&D Systems AF2418), rabbit anti-mouse NFH (1:1000, Encor Biotechnology RPCA-NF-H), chicken anti-mouse NFH (1:1000, Encor Biotechnology CPCA-NF-H), rat anti-mouse iNOS (1:100, ThermoFisher 14-5920-82), mouse anti-human/mouse IL-1β (1:100, Cell Signaling 12242S), rat anti-human/mouse CD45 (1:200, ThermoFisher MA5-17687), rabbit anti-mouse cleaved IL-1β (1:100 Cell Signaling 63124S), Rabbit anti-mouse Arg1 (1:200, Cell Signaling 93668S, rat anti-mouse CD16/32 (1:100, BD Pharmingen 553141). rabbit anti-mouse P22phox (1:200 Cell Signaling 37570S), chicken anti-human/mouse GFAP (1:1000, Biolegend 829401), rabbit anti-human/mouse βAPP (1:200, Thermo Fisher Scientific 36-6900), rabbit anti-human/mouse Ki67 (1:200, Abcam ab15580), and rabbit anti-mouse p16 INK 4 A (1:100, Cell Signaling 29271S). The following secondary antibodies from Jackson ImmunoResearch were used at 1:400 dilution: Alexa Fluor 488 donkey anti-mouse IgM, Alexa Fluor 488 donkey anti-mouse IgG, Cyanine Cy3 donkey anti-chicken IgY, Alexa Fluor 647 donkey anti-rat IgG, Cyanine Cy3 donkey anti-rat IgG, Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 647 donkey anti-rabbit IgG. Mouse spinal cord histology For eriochrome cyanine (EC) and neutral red (NR) visualization of serial spinal cord lesions, spinal cord sections were stained as previously described 26 – 28 . Alternatively, serial spinal cord lesions were also visualized by immunofluorescence labeling of MBP. Brightfield images were then acquired using the Olympus VS110 Slidescanner with a 10x 0.4 NA air objective. Lesion ROIs were drawn based on demyelinated areas in the SCWM that have lower EC staining and higher NR staining and their total areas were quantified using the CEllSens Dimension software (Olympus). Lesion volume was estimated by multiplying the distance separating each serial section (400 µm) by the sum of lesion areas in the serial spinal cord sections from each sample. Labeling tissues for immunofluorescence confocal microscopy As previously described 26 – 28 , slides with mouse spinal cord samples were warmed to room temperature (RT) for 10 min. When MBP staining is required, slides were delipidated by successive wash of 50%, 70%, 90%, 95%, 100%, 95%, 90%, 70%, and 50% ethanol. Then, samples were rehydrated in PBS for 10 min and permeabilized with 0.2% Triton-X100 in PBS for 10 min. Samples were then blocked with donkey blocking solution (PBS, 10% donkey serum, 1% BSA, 0.1% cold fish stain gelation, 0.1% Triton X-100, 0.05% Tween-20) for 1h at RT or overnight at 4 degrees. Alternatively, E06 antibody staining, 5 µg/ml of purified Rat Anti-Mouse CD16/CD32 Fc blocking antibody (5 µg/ml, BD Pharmingen) was added to the blocking buffer. After blocking, samples were incubated with primary antibodies in antibody dilution buffer (PBS, 1% BSA, 0.1% cold fish stain gelation, 0.1% Triton X-100) for overnight incubation at 4 degrees. Samples were then washed 3 times, 5 min each with PBS and 0.2% Tween-20 and incubated with secondary antibodies and 1 µg/ml of DAPI resuspended in the antibody dilution buffer for 1h at RT. For samples with high potential of autofluorescence, slides were also blocked using the TrueBlack Lipofuscin Autofluorescence Quencher (Biotium) in accordance with manufacturer’s instructions. Finally, slides were washed 3 times using PBS with 0.2% Tween-20, 5 min each, and coverslips were mounted onto the slides using Fluoromount-G solution (SouthernBiotech). For post-mortem MS tissue samples, after slides were warmed to RT, they were fixed with 4% paraformaldehyde for 10 min, then washed in PBS for 10 min to remove excess PFA. The remaining steps were the same as the mouse spinal cord samples. Immunofluorescence microscopy Immunofluorescence images were acquired using the Leica TCS Sp8 laser confocal microscope at RT, using the 10x 0.40 NA air object or the 25x 0.5 NA water objective. The 405 nm, 488 nm, 552 nm, and 640 nm lasers were used to excite the fluorophores from antibodies bound to samples and detected by two low dark current Hamamatsu PMT detectors and two high sensitivity hybrid detectors. Images were acquired in 8-bits, in a z-stack using unidirectional scanning, 1 airy unit pinhole, 0.75x zoom, and 0.57 µm optical sections and 2048 x 2048 pixels xy resolution. Alternatively, images were acquired using the Zeiss LSM700 confocal microscope with a 20x 0.8 NA air objective using similar settings. Images were also acquired using the Zeiss Axio Observer 7 widefield microscope with Colibri 7 LED illumination and a 25x 0.85 NA water objective, followed by constrained iterative deconvolution processing. Equal laser, gain, offset, and exposure settings to maximize contrast and minimize saturation were consistently used for all samples within experiment sets. A sample slide stained with only the secondary antibodies and DAPI was used for each experiment to control for non-specific secondary immunofluorescence. Leica Application Suite X or Zeiss Zen Black software was used for image acquisition, ImageJ was used for image threshold and particle analysis. Image analysis Z-stack confocal images of spinal cords were analyzed with ImageJ (Fiji, NIH) as previously described 26 – 28 . Briefly, maximum intensity projections were created for each channel/marker z-stack and converted from 8-bit to RGB. The lesion ROI or equivalent area in the contralateral normal appear white matter (NAWM) was drawn while the area outside the ROI was not analyzed. Positive signal was determined using the color brightness threshold set consistently using a predetermined value by comparing the secondary antibody-stained control and NAWM. Lesion ROIs were drawn based on markers that define the SCWM lesion area such as CD16/32, IBA1, or MBP. The analyze particles function was then used to quantify the positive signals in each ROI. To avoid bias, the same threshold values for setting the positive signal, as well as the size and circularity settings for particle analysis were used for all samples in each experimental set. For representative images shown, maximum intensity projection of each channel/marker in a z-stack were merged and displayed using pseudo colors. Only brightness and contrast settings were adjusted in ImageJ, and consistently between samples for better displaying the images. Statistics and reproducibility Data were collated in Microsoft Excel and graphs were generated using GraphPad Prism 10.1 (LaJolla, CA). Data shown are the individual data points where each point on a graph represents a separate mouse. The mean ± SD are also shown. No sample size calculation was performed. Sample size was determined based on previously published results 26 – 28 and based the cost of experiment, feasibility of the experiment, as well as the availability of sex and aged matched mice. No data was excluded from the analyses. Sample sizes are reported in the figure legends and only one measurement is recorded per sample. Littermate mice were randomly selected for each experimental condition and treatment. Blinding was not conducted. For analysis of statistical significance between the means of two or more treatment groups against the control group, one-way ANOVA with Tukey’s multiple comparison test was used. Two-tailed, unpaired t-test was used to compared data with only two groups. Kolmogorov-Smirnov test was used to verify the normal distribution of data. Specific P-values are reported in each figure where results are considered statistically significant where p < 0.05. Declarations Data availability All data are available upon reasonable request. ACKNOWLEDGEMENTS Y.D. acknowledges operating grant support from the University of Saskatchewan College of Medicine, the Natural Sciences and Engineering Research Council of Canada, MS Canada, Saskatchewan Health Research Foundation, and Brain Canada Foundation. A.S. and J.P. acknowledge undergraduate Biomedical Project research support from the College of Medicine, University of Saskatchewan. J.P. and K.T. also acknowledge undergraduate student research award support from the Natural Sciences and Engineering Research Council of Canada. A.P. holds a Tier 1 senior Canada Research Chair in Multiple Sclerosis and is funded by the CIHR, MS Canada, the International Progressive MS Alliance, the Canadian Foundation for Innovation and the National MS Society (USA). We also thank Histology and Cancer Cluster Core Facilities at the University of Saskatchewan, as well as the Hotchkiss Brain Institute Advanced Microscopy Platform Facility at the University of Calgary, for technical help. AUTHOR CONTRIBUTIONS Y.D. conceived the project and designed experiments. R.Y., B.M.L., Y.D., A.S., and K.T performed experiments and analyzed data. D.K.K. provided support and data for EAE experiments. S.Z., W.K., S.L, and A.P. provided P-MS brain specimens and their characterization. S.T. and J.L.W. provided critical reagent support. Y.D. supervised the overall study and wrote the manuscript. All authors reviewed and edited the manuscript. COMPETING INTERESTS The authors declare no competing interests. References Yong, H.Y.F. & Yong, V.W. Mechanism-based criteria to improve therapeutic outcomes in progressive multiple sclerosis. Nat Rev Neurol 18, 40–55 (2022). University of California, S.F.M.S.E.T., et al. 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Dong, Y., Lozinski, B.M., Silva, C. & Yong, V.W. Studying the microglia response to oxidized phosphatidylcholine in primary mouse neuron culture and mouse spinal cord. STAR Protoc 2, 100853 (2021). Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedFigures.docx Cite Share Download PDF Status: Published Journal Publication published 01 Dec, 2025 Read the published version in Nature Neuroscience → 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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Diego","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Witztum","suffix":""},{"id":334827526,"identity":"a5e1b1bc-cdd3-4afe-b45a-f988c784b46d","order_by":11,"name":"Alexandre Prat","email":"","orcid":"https://orcid.org/0000-0001-6188-0580","institution":"The Research Center of the Centre Hospitalier de l’Université de Montréal","correspondingAuthor":false,"prefix":"","firstName":"Alexandre","middleName":"","lastName":"Prat","suffix":""},{"id":334827514,"identity":"8272e96f-e59a-4590-9506-2886e9d68ac4","order_by":12,"name":"Yifei Dong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACxgbGBx8Y2IAs9gYQP4EYLcyGM8BaeA4QqYWBAaZFIoFILcztzYwNDGU2efKRb4w//PiTxsDffoCAw3oOA7WcSys2vJ1jJtnblsMgcYaATYwz8o8/YGw7nLhxdo4ZA29DBYMBIccxzn8MDDSQlplnjD/++QPUwv+AkC3MEC3zJXgMpHnYchgMJAjZ0pPM2JBwLi1xA09ambRsWxqPxA0Cthi2A73/ocwmcX774c0f3/xJluPvJ2CLYQMDJDIMDkAEePCrBwJ5OKOBoNpRMApGwSgYqQAAARpFXb8tNtgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1483-2046","institution":"University of Saskatchewan","correspondingAuthor":true,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2024-07-24 04:35:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4792293/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4792293/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41593-025-02113-y","type":"published","date":"2025-12-01T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61730397,"identity":"a528cb5a-2629-458b-8978-679bfd87429a","added_by":"auto","created_at":"2024-08-05 00:24:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2938435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxPC deposition induces chronic demyelination and neuroinflammation in the CNS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Schematic of experimental design comparing acute and chronic OxPC lesions in the SCWM. b-d) Representative immunofluorescence images of the day 7 and day 42 OxPC lesions and the respective contralateral NAWM labeled with DAPI (blue), IBA1 (red), OLIG2 (green), and MBP (grey) (b), or the day 7 and day 42 lesions labeled with DAPI (blue), CD11b (red), P22phox (green) and GFAP (grey) (c), or with DAPI (blue), CD11b (red), and IL-β (green) (d).\u0026nbsp; e-n) Graphs comparing the average lesion epicenter size (e), the amount of CD11b (f) and IBA1 (g), the average size of IBA1\u003csup\u003e+ \u003c/sup\u003ecells (h), the density of OLIG2\u003csup\u003e+ \u003c/sup\u003ecells (i), the amount of MBP (j) and P22phox (k), the proportion of CD11b\u003csup\u003e+ \u003c/sup\u003ecells that overlap with P22phox (l), the amount of IL-1β (m), and the proportion of CD11b\u003csup\u003e+ \u003c/sup\u003ecells that overlap with IL-1β (n), between day 7 acute and day 42 chronic OxPC lesions. Data acquired from 2 experiments with 4 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test, comparing day 7 and 42 lesions. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/fd289427cad01da0e8f97e0c.png"},{"id":61730404,"identity":"7be9de7b-ead6-47c8-bc7f-0b711b95e399","added_by":"auto","created_at":"2024-08-05 00:24:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3485495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxPC mediates endogenous lipid peroxidation and chronic neurodegeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-d) Representative immunofluorescence images of the day 7 and day 42 OxPC lesions and the respective contralateral NAWM labeled with DAPI (blue), NFH (red), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green), and CD11b (grey) (a), or the day 7 and day 42 lesions labeled with DAPI (blue), NFH (red), and βAPP (green) (b), or with DAPI (blue), CD11b (red), Ki67 (green) and GFAP (c), or with DAPI (blue), IBA1 (red), and pH2A.X (green).\u0026nbsp; e-h) Graphs comparing the amount of E06\u003csup\u003e+ \u003c/sup\u003eendogenously formed OxPC (e), the relative difference in lesional NFH\u003csup\u003e+ \u003c/sup\u003epercent area (f), the average size of NFH\u003csup\u003e+ \u003c/sup\u003eaxons (g), and the overlap of NFH\u003csup\u003e+ \u003c/sup\u003eaxons with βAPP (h), between day 7 and day 42 OxPC lesions. i) Heatmap showing the distribution of βAPP across the day 7 or day 42 OxPC lesions. j-m) Graphs comparing the density of CD11b\u003csup\u003e+ \u003c/sup\u003eKi67\u003csup\u003e+ \u003c/sup\u003ecells (j), the proportion of CD11b\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with Ki67 (k), the amount of pH2A.X (l), and the amount of IBA1\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with pH2A.X (m) between day 7 and day 42 OxPC lesions. Data acquired from 2 experiments with 4 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test, comparing day 7 and 42 lesions. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/2025c5109aa03d6a6a08224b.png"},{"id":61730396,"identity":"f43fe3f1-1940-44be-8625-efc0bbbcf0ac","added_by":"auto","created_at":"2024-08-05 00:24:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2180551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparing and fate mapping microglia in chronic OxPC and LPC lesions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Schematic of experimental design comparing OxPC and LPC induced chronic lesions in the SCWM of CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom \u003c/sup\u003emice. b-d) Representative immunofluorescence images of day 42 OxPC lesions and LPC lesions labeled with DAPI (blue), tdTomato (red), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green), and IBA1 (grey) (b), or with tdTomato (red), OLIG2 (green) and MBP (grey) (c), or with DAPI (blue), tdTomato (red), and NFH (green) (d).\u0026nbsp; e-m) Graphs comparing the average lesion epicenter size (e), the amount of IBA1 (f) and E06\u003csup\u003e+ \u003c/sup\u003eOxPC (g), the proportion of IBA1\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with tdTomato (h), the percent of tdTomato\u003csup\u003e+\u003c/sup\u003e cells that were larger than 100 µm\u003csup\u003e2 \u003c/sup\u003ein size (i), the average size of tdTomato\u003csup\u003e+ \u003c/sup\u003ecells that were larger than 100 µm\u003csup\u003e2\u003c/sup\u003e (j), the amount of MBP (k), the density of OLIG2\u003csup\u003e+ \u003c/sup\u003ecells (l), and the density of NFH\u003csup\u003e+ \u003c/sup\u003eaxons (m), between day 42 OxPC lesions and day 42 LPC lesions. Data acquired from 2 experiments with 2 to 3 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test, comparing OxPC and LPC lesions. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/7c0e5357303b4998bfac64de.png"},{"id":61730399,"identity":"17ede56d-d3ae-4fc5-b8f2-03009c267293","added_by":"auto","created_at":"2024-08-05 00:24:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2588531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAging exacerbates OxPC mediated chronic neurodegeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Schematic of experimental design comparing chronic OxPC lesions in 6wk and 52k mice. b) Representative bright field images of serial spinal cord section from 6wk and 52wk mice showing the extent of day 42 OxPC lesions. c-e) Graphs comparing the spread of the OxPC lesion (c), the average size of lesion epicenter (d), the estimated lesion volume (e), between 6wk and 52wk mice at day 42. f) Graph comparing the number of serial spinal cord sections containing lesions at acute (day 3 and 7) and chronic (day 42) time points between 6wk and 52wk mice. g-i) Representative immunofluorescence images of day 42 OxPC lesions in 6wk and 52wk mice labeled with DAPI (blue), IBA1 (red) and E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green) (g), or with NFH (red), OLIG2 (green) and MBP (grey) (h), or with NFH (red), βAPP (green), and CD16/32 (i). j-r) Graphs comparing the amount of E06\u003csup\u003e+ \u003c/sup\u003eOxPC (j), the amount of IBA1 (k) and IBA1\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with E06\u003csup\u003e+ \u003c/sup\u003eOxPC (l), the density of OLIG2\u003csup\u003e+ \u003c/sup\u003ecells (m), the amount of MBP (n), the density of NFH\u003csup\u003e+ \u003c/sup\u003eaxons (o) and their average size (p), the amount of βAPP (q), and the proportion of NFH\u003csup\u003e+ \u003c/sup\u003eaxons that overlap with βAPP (r), between day 42 chronic OxPC lesions from 6wk and 52wk mice. Data acquired from 2 experiments with 4 or 5 mice per group per experiment for day 42 OxPC lesions. For day 3 and day 7 lesion in (f), data was acquired from 2 experiments with 3 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test, comparing day 42 lesions from 6wk and 52wk mice. For comparing lesion spread between 6wk and 52wk mice across days 3, 7, and 42 in (f), ordinary two-way ANOVA with Tukey’s multiple comparison test was performed. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/df160b54303319a08fa6103a.png"},{"id":61730524,"identity":"dae7807e-4905-47ab-aab9-7c9f5d5d9ea9","added_by":"auto","created_at":"2024-08-05 00:32:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1981520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAging dysregulates microglia and monoMac responding to chronic neurodegeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea-b) Representative immunofluorescence images of chronic OxPC lesions in 6wk and 52wk mice labeled with CD11b (red), P22phox (green) and GFAP (grey) (a), or with DAPI (blue), IBA1 (red) and IL-1β (green) (b). c-g) Graphs comparing the amount of CD11b (c), the amount of P22phox (d) and CD11b\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with P22phox (e), the amount of IL-1β (f) and IBA1\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with IL-1β (g), between day 42 OxPC lesion from 6wk and 52wk mice. Data was acquired from 2 experiments with 4 or 5 mice per group per experiment. h) Schematic of experimental design comparing OxPC chronic lesions in the SCWM between 6wk and 52wk CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom \u003c/sup\u003emice. i) Representative immunofluorescence images of chronic OxPC lesions in 6wk and 52wk CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom \u003c/sup\u003emice labeled with tdTomato (red), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green) and IBA1 (grey). l-n) Graph comparing the amount of tdTomato (l), the proportion of IBA1\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with tdTomato (m), and the proportion of E06\u003csup\u003e+ \u003c/sup\u003eOxPC that overlapped with tdTomato\u003csup\u003e+ \u003c/sup\u003ecells (n). Data was acquired from 2 experiments with 2 or 3 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test, comparing day 42 lesions from 6wk and 52wk mice. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/d84c67cbe716c45c338f02cd.png"},{"id":61730525,"identity":"8477d711-86d2-4859-bb41-05f401dd85d8","added_by":"auto","created_at":"2024-08-05 00:32:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2546704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroglia loss during chronic phase of disease exacerbates chronic neurodegeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Schematic of experimental design comparing chronic neurodegeneration in the presence of absence of microglia depletion in CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Rosa26\u003csup\u003eiDTR+ \u003c/sup\u003emice. b) Graph comparing the survival of CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Rosa26\u003csup\u003eiDTR+ \u003c/sup\u003emice treated with PBS or DT during the acute phase (days 0-7) or chronic phase (days 35-42) of disease. c) Graph comparing chronic OxPC lesion volume in days 35-42 PBS or DT treated mice. d-g) Representative immunofluorescence images of chronic OxPC lesions from days 35-42 PBS or DT treated mice labeled with DAPI (blue), IBA1 (red) and E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green) (d), or with CD11b (red), P22phox (green) and GFAP (grey) (e), or with DAPI (blue), IBA1 (red), and IL-1β (green) (f), or with NFH (red), OLIG2 (green), and MBP (grey) (g). h-r). Graphs comparing the amount of IBA1 (h), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (i) and their overlap with IBA\u003csup\u003e+ \u003c/sup\u003ecells (j), the amount of CD11b (k), the amount of P22phox (l) and its overlap with CD11b\u003csup\u003e+ \u003c/sup\u003ecells (m), the amount of IL-1β (n) and its overlap with IBA1\u003csup\u003e+ \u003c/sup\u003ecells (o), the amount of MBP (p), the density of NFH\u003csup\u003e+ \u003c/sup\u003eaxons (q), and the density of OLIG2\u003csup\u003e+ \u003c/sup\u003ecells (r), between chronic OxPC lesions from days 35-42 PBS or DT treated mice. Data acquired from 2 experiments with 3 or 4 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/ff75eb26d573c10e672c562d.png"},{"id":61730403,"identity":"bce453c8-9c9c-4d4f-be03-312b3568f3df","added_by":"auto","created_at":"2024-08-05 00:24:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2369645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCAS1/4 deficiency reduces chronic OxPC generation and neurodegeneration.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Representative immunofluorescence images of serial spinal cord sections from CAS1/4\u003csup\u003e+/+ \u003c/sup\u003eor CAS1/4\u003csup\u003e-/- \u003c/sup\u003emice with chronic OxPC lesions at day 42, labeled with MBP (grey). b-d) Representative immunofluorescence images showing day 42 chronic OxPC lesions from CAS1/4\u003csup\u003e+/+ \u003c/sup\u003eor CAS1/4\u003csup\u003e-/- \u003c/sup\u003emice labeled with DAPI (blue), cleaved IL-1β (green), and CD45 (red) (b), or with DAPI (blue), IBA1 (red), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green), and GFAP (grey) (c), or with DAPI (blue), NFH (red), and MBP (grey) (d). e-m) Graphs comparing the estimated lesion volume (e), lesion epicenter area (f), the amount of CD45 (g), cleaved IL-1β (h), CD45 that overlapped with cleaved IL-1β (i), IBA1 (j), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (k), and MBP (l), as well as NFH\u003csup\u003e+ \u003c/sup\u003eaxon density (m) between chronic OxPC lesions from CAS1/4\u003csup\u003e+/+ \u003c/sup\u003eor CAS1/4\u003csup\u003e-/- \u003c/sup\u003emice. Data acquired from 2 experiments with 4 or 5 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test. Data are represented as mean ± SD.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/f9e2c403e16506615f04d6b8.png"},{"id":61730526,"identity":"efe04077-4f54-4b57-bedb-8c665dba67c9","added_by":"auto","created_at":"2024-08-05 00:32:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2353813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-1R1 blockade ameliorates OxPC mediated chronic neurodegeneration and IL-1β is associated with OxPC accumulation in CAL from P-MS.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea) Schematic of experimental design comparing chronic neurodegeneration with or without anakinra blockade of IL-1R1 from days 28-42. b-c) Representative immunofluorescence images showing day 42 chronic OxPC lesions from mice treated with PBS or anakinra (Anak) from days 28-42 labeled with DAPI (blue), IBA1 (red), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green), and GFAP (grey) (b), or with DAPI (blue), NFH (red), OLIG2 (green), and MBP (grey) (c). d-i) Graphs comparing the fold difference in the amount of E06\u003csup\u003e+ \u003c/sup\u003eOxPC (d), IBA1 (e) and the of IBA1\u003csup\u003e+ \u003c/sup\u003ecells that overlapped with E06\u003csup\u003e+ \u003c/sup\u003eOxPC (f), the amount of MBP (g), the density of OLIG2\u003csup\u003e+ \u003c/sup\u003ecells (h), and the density of NFH\u003csup\u003e+ \u003c/sup\u003eaxons (i), between chronic OxPC lesions from PBS or Anak treated\u003csup\u003e \u003c/sup\u003emice. Data acquired from 2 experiments with 3 or 4 mice per group per experiment. Statistical significance reported as p-values, two-tailed, unpaired t-test. j) Representative bright field images of post-mortem P-MS brain tissue sections containing CAL, 4x4 grids shows areas where immunofluorescence images were acquired to compare between CAL and NAWM. k) Representative immunofluorescence images showing P-MS brain labeled with DAPI (blue), CD45 (red), E06\u003csup\u003e+ \u003c/sup\u003eOxPC (green), and IL-1β (grey). l-q) Graphs comparing the amount of CD45 (l), the fold difference in CD45\u003csup\u003e+ \u003c/sup\u003eaverage cell size (m), the amount of E06\u003csup\u003e+ \u003c/sup\u003eOxPC (n) and its overlap with CD45\u003csup\u003e+ \u003c/sup\u003ecells (o), the amount of IL-1β (p) and its overlap with CD45\u003csup\u003e+ \u003c/sup\u003ecells (q), between CAL and NAMW from P-MS brains. Data acquired from the brains of seven individuals with P-MS, 2 FOVs containing CAL rim and 1 FOV of the NAWM were analyzed for each brain. Statistical significance reported as p-values, two-tailed, paired t-test.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/3f2256c3ba672946734a0835.png"},{"id":97223979,"identity":"084a1a02-e639-4486-b63c-1d453546da90","added_by":"auto","created_at":"2025-12-02 08:07:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":20661193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/3f857892-713e-4613-8234-6334a4cf93a6.pdf"},{"id":61730401,"identity":"2c43d4cf-43b0-46ae-a7ba-a9dc5d87ce41","added_by":"auto","created_at":"2024-08-05 00:24:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4820709,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4792293/v1/2e1a269be508e39081b88c80.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Oxidized phosphatidylcholines induce chronic neurodegeneration partly through IL-1β mediated positive feedback","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eChronic neurodegeneration defined as the continuous degradation of neuronal structure and function, as well as neuronal death, is significantly associated with disability accumulation and mortality in people living with progressive multiple sclerosis (P-MS)\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. Compared to relapsed-remitting MS in which disease activity is largely driven by peripheral immune cells that infiltrate the CNS\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, formation of new lesions associated with blood-brain barrier damage is less frequent in P-MS. Instead, there is an increased number of mixed active-inactive/chronic active lesions (CAL)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, which are characterized by a relatively inactive lesion core surrounded by a radially expanding rim with reactive microglia/macrophages, demyelination, and neuroaxonal loss\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. MRI and post-mortem analysis indicate CAL are sites of chronic neurodegeneration that enlarge over months to years, and they are associated with worsening disability and MS progression\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. However, because the molecular mechanisms that promote chronic neurodegeneration and CAL expansion are relatively unknown, developing effective treatments against disease progression and disability accumulation in P-MS remains challenging\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe elevation of oxidative stress byproducts such as oxidized phosphatidylcholines (OxPC) with degenerating neuronal cells and axons in P-MS\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and the accumulation of reactive microglia/macrophages with phenotypes associated with inflammation and free radical production at the rim of expanding CAL\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\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 suggest that oxidative injury contributes to chronic neurodegeneration in P-MS. We previously demonstrated OxPC found in relapse-remitting MS lesions mediate acute neuroinflammation and neurodegeneration in the spinal cord white matter (SCWM) of young and middle-aged mice\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, suggesting OxPC may be key mediators of tissue injury in MS. Yet, how OxPC affect chronic neurodegeneration in P-MS is not known because commonly used rodent models of MS express relatively low levels of OxPC compared to human MS\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Given OxPC are implicated as key mediators of tissue degeneration in chronic non-CNS diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e and that they accumulate in brain lesions throughout different stages of MS progression\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and even in deep GM where neurodegeneration commonly occurs\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, it is imperative to investigate the role of OxPC in P-MS. Here, we aimed to determine the potential of acute OxPC injuries progressing to mediate chronic neurodegeneration in the mouse SCWM. Our results highlight that OxPC deposition uniquely promotes a chronic lesion with compartmentalized neuroinflammatory and neurodegenerative features similar to CAL in P-MS. Notably, we found aging, microglia, and IL-1β/IL-1 receptor type I (IL-1R1) signaling are key factors that modulate the intensity of OxPC mediated chronic neurodegeneration.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOxPC induce chronic neuroinflammation, demyelination, and neurodegeneration\u003c/h2\u003e \u003cp\u003ePrevious imaging mass spectrometry experiments indicate that 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) is one of the most abundant species of OxPC in active MS lesions and that its deposition in the CNS mediates acute neuroinflammation, demyelination, and neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To determine if acute focal lesions induced by POVPC progress to become sites of chronic neurodegeneration akin to CAL in P-MS, we first induced lesions in the SCWM of 6-week-old mice by a one-time stereotactic injection of POVPC and compared tissue pathology after 7 and 42 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Unlike the experimental autoimmune encephalomyelitis (EAE) model of MS neuroinflammation where lesions form stochastically and are therefore difficult to assess over time\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and the lysolecithin (LPC) model of MS focal demyelination where remyelination and repair occurs relatively quickly\u003csup\u003e\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, the focal lesion induced by POVPC remained chronically inflammatory and demyelinated after 42 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). While day 42 chronic lesions were smaller in area compared to day 7 acute lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), scar forming glial fibrillary acidic protein (GFAP)\u003csup\u003e+\u003c/sup\u003e astrocytes at the lesion border and the intralesional accumulation of CD11b\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes were similar between chronic and acute lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, F). The lack of myelin basic protein (MBP) in day 42 lesions also indicates remyelination failure despite an increase in oligodendrocyte transcription factor (OLIG2)\u003csup\u003e+\u003c/sup\u003e oligodendroglia\u003csup\u003e37\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, I, J). Interestingly, the percent area of lesional IBA1\u003csup\u003e+\u003c/sup\u003e cells and the average size of these cells were reduced at day 42 compared to day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, G, H), suggesting that microglia/macrophage composition or behavior is different in chronic lesions compared to acute lesions. Indeed, the levels of lesional P22phox subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and interleukin (IL)-β, as well as the proportion of CD11b\u003csup\u003e+\u003c/sup\u003e that overlapped with these two inflammatory molecules, were elevated at day 42 compared to day 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, K-N).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOxPC can be specifically bound and detected by the natural IgM antibody E06\u003csup\u003e29, 38\u003c/sup\u003e. Since we previously found minimal E06\u003csup\u003e+\u003c/sup\u003e immunoreactivity in acute lesions at 24 h post POVPC injection\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, the elevation of E06\u003csup\u003e+\u003c/sup\u003e immunoreactivity thereafter represents the accumulation of endogenously produced OxPC. Thus, similar levels of E06\u003csup\u003e+\u003c/sup\u003e immunoreactivity in day 7 and day 42 lesions suggested that the accumulation of endogenous OxPC was sustained in chronic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, E). To assess neurodegeneration in chronic OxPC lesions, we compared neurofilament heavy chain (NFH)\u003csup\u003e+\u003c/sup\u003e axons between day 7 and day 42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Unlike NFH\u003csup\u003e+\u003c/sup\u003e axons in the normal appearing white matter (NAWM), axons within day 7 lesions appear enlarged and were more dispersed across the lesion, which suggested edema and axonal stress. In contrast, NFH\u003csup\u003e+\u003c/sup\u003e axons are mostly absent in the core of day 42 lesions, which suggests neurodegeneration had occurred; axons that remain are largely found near the lesion edges and are similar in size as axons in the NAWM. The significant decrease of lesional NFH\u003csup\u003e+\u003c/sup\u003e area relative to NAWM NFH\u003csup\u003e+\u003c/sup\u003e area and average NFH\u003csup\u003e+\u003c/sup\u003e axon size in day 42 lesions compared to day 7 lesions supports these observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). Furthermore, the proportion of NFH\u003csup\u003e+\u003c/sup\u003e axons that overlapped with beta amyloid precursor protein (βAPP), a marker of axonal injury\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, trended to be higher in chronic lesions compared to acute lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Additionally, while βAPP\u003csup\u003e+\u003c/sup\u003e NFH\u003csup\u003e+\u003c/sup\u003e axons were found mostly at lesion cores at day 7, they were found more at the lesion edges by day 42 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, I), which suggests an outward expansion of axonal injury from the lesion core. Interestingly, compared to acute lesions, chronic lesions also had significantly less CD11b\u003csup\u003e+\u003c/sup\u003e Ki67\u003csup\u003e+\u003c/sup\u003e proliferative cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, J, K) but significantly more IBA1\u003csup\u003e+\u003c/sup\u003e cells that were labeled with phosphorylated histone (pH2A.X) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, L, M), which is a marker of DNA damage and repair\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. These observations are additional evidence that mononuclear phagocytes found in chronic lesions were in distinct cellular states compared to those found in acute lesions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCollectively, these results indicated that the deposition of MS-relevant OxPC, such as POVPC, in the CNS induced a CAL-like focal lesion with endogenous OxPC accumulation as well as compartmentalized chronic neuroinflammation, demyelination, and neurodegeneration. Notably, OxPC induced chronic lesions remained inflamed and unrepaired after 100 days (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eOxPC induced chronic neurodegeneration features endogenous lipid peroxidation and reemergence of monocyte derived macrophages (monoMac)\u003c/h2\u003e \u003cp\u003eReactive microglia/macrophages found in CAL associate with disease activity and lesion expansion in P-MS\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, the differential contribution of microglia versus monoMac to CAL disease activity and MS progression remains difficult to study. To address this knowledge gap, we next utilized the chronic OxPC lesions to interrogate the ontogeny of the mononuclear phagocytes during chronic neurodegeneration using CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice. These mice were first treated with tamoxifen 3 weeks before the experiment to label long-lived microglia with the tdTomato fluorescent reporter protein and to allow for the short-lived tdTomato labeled monocytes to turnover. POVPC was then injected into the mouse SCWM and the proportion of microglia (IBA1\u003csup\u003e+\u003c/sup\u003e tdTomato\u003csup\u003e+\u003c/sup\u003e) versus monoMac (IBA1\u003csup\u003e+\u003c/sup\u003e tdTomato\u003csup\u003e\u0026minus;\u003c/sup\u003e) was analyzed in chronic lesions at day 42. Additionally, separate groups of CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice were injected with an equivalent amount of LPC to determine how chronic OxPC lesions differ from the LPC induced focal demyelination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). By day 42, the overall size of the chronically demyelinated lesion area marked by MBP disruption (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-E, K) and the amount of total lesional IBA1\u003csup\u003e+\u003c/sup\u003e microglia/macrophage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) were similar between OxPC and LPC injected mice. Unlike day 7 acute OxPC\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e and LPC lesions\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e where \u0026gt;\u0026thinsp;90% of lesional macrophages are tdTomato\u003csup\u003e+\u003c/sup\u003e microglia, only approximately 50% of IBA1\u003csup\u003e+\u003c/sup\u003e cells from chronic OxPC and LPC lesions overlapped with tdTomato (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH), which suggested an increase contribution of monoMac to the total macrophage population within chronic focal lesions in the CNS. Interestingly, IBA\u003csup\u003e+\u003c/sup\u003e tdTomato\u003csup\u003e+\u003c/sup\u003e microglia in OxPC lesion were significantly larger in size compared to microglia in LPC lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J), suggesting that microglia in chronic OxPC lesions may be more representative of enlarged/amoeboid reactive microglia found in CAL from P-MS\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMore importantly, significant endogenous OxPC deposition was only found in chronic OxPC lesions but not in chronic LPC lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, G) and OxPC was only minimally detected near the meninges of spinal cord and cerebellum lesions from day 45 chronic EAE mice (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Furthermore, the remyelinating potential and chronic neurodegeneration marked by OLIG2\u003csup\u003e+\u003c/sup\u003e oligodendroglia accumulation and NFH\u003csup\u003e+\u003c/sup\u003e axon loss, respectively, were significantly worse in chronic OxPC lesions compared to chronic LPC lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL, M). Thus, endogenous lipid peroxidation, chronic neurodegeneration, and the equal accumulation of reactive microglia and monoMac are unique pathological features of chronic OxPC lesions that may be informative for CAL expansion and P-MS.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAging exacerbates OxPC induced chronic neurodegeneration\u003c/h3\u003e\n\u003cp\u003eAging is a major risk factor for neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e and P-MS onset most often occurs during middle age\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. To investigate how aging changes the susceptibility of the CNS to OxPC mediated chronic neurodegeneration, we induced OxPC deposition in the SCWM 6wk old young mice and 52wk old middle-aged mice and analyzed chronic neurodegeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Compared to 6wk old mice, day 42 chronic lesions in 52wk old mice had greater lesion spread and significantly greater total lesion volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-E). While there was a significant reduction in lesion spread at day 42 compared to day 7 in 6wk old mice, this did not occur in 52wk old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), suggesting that the response to limit OxPC mediated chronic neurodegeneration became less effective in the aging CNS. Indeed, there was significantly greater accumulation of endogenous E06\u003csup\u003e+\u003c/sup\u003e OxPC as well as OxPC that overlapped with IBA1\u003csup\u003e+\u003c/sup\u003e microglia/macrophages in 52wk old chronic lesions compared to 6wk old chronic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, J-L). Moreover, while there were no differences in IBA1\u003csup\u003e+\u003c/sup\u003e microglia/macrophages, OLIG2\u003csup\u003e+\u003c/sup\u003e oligodendroglia, and overall amount of MBP between chronic lesions from 6wk old and 52wk old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, M, N), the density of surviving NFH\u003csup\u003e+\u003c/sup\u003e axons in lesions from middle-aged mice were significantly lower than axons in lesions from young mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, O). In addition, the average size of NFH\u003csup\u003e+\u003c/sup\u003e axons, the level of βAPP, and the proportion of NFH\u003csup\u003e+\u003c/sup\u003e axons that overlap with βAPP\u003csup\u003e+\u003c/sup\u003e in chronic lesions from 52wk old mice were significantly greater compared to chronic lesions from 6wk old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, P-R). These observations indicate that aging exacerbates chronic neurodegeneration and endogenous OxPC accumulation in the CNS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAging promotes microglial dysfunction during OxPC induced chronic neurodegeneration\u003c/h2\u003e \u003cp\u003eWe previously found that compared to 6wk old mice, microglia/macrophages from 52wk old mice had significant transcriptional and functional reprogramming in response to acute OxPC mediated neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To investigate if aging also causes microglial dysfunction during chronic OxPC mediated neurodegeneration, we further profiled the functional phenotype of microglia/macrophages found in day 42 lesions of 6wk old and 52wk old mice. While there was no difference in total IBA1\u003csup\u003e+\u003c/sup\u003e cells between young and middle-aged lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK), there was greater accumulation of CD11b\u003csup\u003e+\u003c/sup\u003e cells in middle-aged chronic lesions compared to young lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, C), suggesting that aging may alter microglia/macrophage composition in chronic lesions. In addition, P22phox and IL-1β, which were upregulated by microglia/macrophages in day 42 chronic lesions compared to day 7 acute lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK-N), were also significantly elevated in middle-aged lesions compared to young lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-G). Middle-aged lesions also had significantly greater amount of p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003ea\u003c/sup\u003e (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, D), a marker of senescence in the aging CNS\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In contrast, the expression of induced nitric oxide synthase (iNOS) and arginase 1 (Arg1) were relatively low in chronic lesions and there were no differences between young and middle-aged mice (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C, E-H). These observations suggest that aging promoted inflammatory dysregulation in microglia/macrophages during chronic neurodegeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe composition of macrophages in day 42 chronic OxPC lesions was approximately 50% microglia and 50% monoMac (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH) whereas macrophages in day 7 acute lesions were \u0026gt;\u0026thinsp;90% microglia\u003csup\u003e27\u003c/sup\u003e. Since increased engraftment of monoMac in the CNS associates with aging and neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, we next analyzed day 42 chronic OxPC lesions in tamoxifen treated 6wk old and 52wk old CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) to investigate whether aging promotes greater replacement of microglia by monoMac in chronic OxPC lesions. Like 6wk old and 52wk old wildtype mice, there was no difference in the amount of IBA1\u003csup\u003e+\u003c/sup\u003e cells between 6wk old and 52wk old CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI, J). However, the abundance of IBA1\u003csup\u003e+\u003c/sup\u003e tdTomato\u003csup\u003e+\u003c/sup\u003e microglia were significantly reduced in 52wk old CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice compared to 6wk old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eL, M). Furthermore, despite increased OxPC accumulation in chronic lesions of 52wk old CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice compared to 6wk old mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eK), the proportion of E06\u003csup\u003e+\u003c/sup\u003e OxPC that overlapped with tdTomato\u003csup\u003e+\u003c/sup\u003e microglia was significantly lower in middle-aged lesions compared to young lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). Together, these results suggest aging facilitated greater monoMac accumulation and reduced microglial sequestration of OxPC in chronic OxPC lesions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMicroglia depletion in chronic OxPC lesions worsens neurodegeneration\u003c/h2\u003e \u003cp\u003eAlthough post-mortem analysis of CAL found in P-MS brains highlight the involvement of reactive, proinflammatory microglia/macrophages\u003csup\u003e9, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e, the pathological role of microglia within CAL and P-MS remains less well understood. Since OxPC deposition in the CNS induces a CAL-like focal lesion with both microglia and monoMac, we next utilized this model to study the functional significance of microglia during chronic oxidative damage, neuroinflammation, and neurodegeneration. Specifically, CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Rosa26\u003csup\u003eiDTR\u003c/sup\u003e mice were treated with tamoxifen to induce diphtheria toxin receptor (DTR) expression in long-lived microglia and to allow short-lived DTR\u003csup\u003e+\u003c/sup\u003e monocytes to turnover. Three weeks later, the SCWM of these mice were then injected with POVPC to induce the focal OxPC lesion, followed by PBS or diphtheria toxin (DT) treatment once every other day between days 0\u0026ndash;7 or 35\u0026ndash;42 after OxPC deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We aimed to compare how DT mediated microglia depletion during the acute phase of the lesion (days 0\u0026ndash;7) or during the chronic phase of the lesion (days 35\u0026ndash;42) differentially impacted the outcome of chronic neurodegeneration at day 42. Unexpectedly, mice with DT treatment during acute neurodegeneration (days 0\u0026ndash;7) failed to survive past day 18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), perhaps due to worsening of acute neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e or non-specific long-term toxicity from DT\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Thus, how the acute microglial response to OxPC deposition regulated the pathology of the chronic lesion could not be determined.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor mice with DT treatment during chronic neurodegeneration (days 35\u0026ndash;42), there was no difference in overall lesion volume by day 42 compared to mice treated with PBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). However, within the lesions, DT treated mice had significantly reduced amount of total IBA1\u003csup\u003e+\u003c/sup\u003e cells compared to PBS treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, H), indicative of microglial depletion. While there was no difference between PBS and DT treated mice in the amount of endogenous E06\u003csup\u003e+\u003c/sup\u003e OxPC deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI), the overlap of E06\u003csup\u003e+\u003c/sup\u003e OxPC with IBA1\u003csup\u003e+\u003c/sup\u003e cells was significantly less in lesions from DT treated mice compared to lesions from PBS treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ), suggesting reduced OxPC clearance by microglia. Interestingly, the overall lesional accumulation of CD11b\u003csup\u003e+\u003c/sup\u003e cells within PBS and DT treated mice was not significantly different (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, K), suggesting that monoMacs may have replaced microglia in DT treated mice. Moreover, while the level of lesional P22phox and the proportion of CD11b\u003csup\u003e+\u003c/sup\u003e cells that overlapped with P22phox were similar between PBS treated and DT treated mice, the level of IL-1β and the proportion of IBA1\u003csup\u003e+\u003c/sup\u003e cells that overlapped with IL-1β were significantly elevated in DT treated mice compared to PBS treated mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, L-O). Furthermore, DT treatment significantly reduced the amount of lesional MBP as well as the density of NFH\u003csup\u003e+\u003c/sup\u003e axons and OLIG2\u003csup\u003e+\u003c/sup\u003e oligodendroglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, P-R) in lesions compared to PBS treatment. Collectively, these results showed that the depletion of microglia in chronic OxPC lesions abnormally elevated IL-1β, impaired remyelination potential, and exacerbated neurodegeneration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eProgressive MS and severity of chronic neurodegeneration associate with OxPC accumulation and IL-1β activity\u003c/h2\u003e \u003cp\u003eThus far, we demonstrated that POVPC deposition in the SCWM of mice induces a CAL-like unremitting lesion with endogenous OxPC generation as well as chronic neuroinflammation and neurodegeneration, but the endogenous mechanism that drive this chronic pathology remain unclear. Interestingly, OxPC can promote IL-1β secretion by myeloid cells including macrophages\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and we previously found IL-1β deposition in the SCWM of mice induced acute endogenous OxPC formation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Since total IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eM) and cleaved IL-1β (Extended Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D) were elevated in chronic OxPC lesions compared to acute lesions, and that IL-1β was further dysregulated when OxPC mediated chronic neurodegeneration was exacerbated by aging (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF) or by microglia depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN, O), we hypothesized that OxPC deposition may promote a vicious cycle of chronic neuroinflammation and oxidative stress at least in part through aberrant IL-1β production and/or signaling. Since caspase 1/4 (CAS1/4) deficiency prevents the processing of pro-IL-β to its active form\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, we tested this hypothesis by inducing chronic OxPC lesions in CAS1/4\u003csup\u003e+/+\u003c/sup\u003e and CAS1/4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice. Although the size of the lesion epicenters at day 42 were similar between CAS1/4\u003csup\u003e+/+\u003c/sup\u003e and CAS1/4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, the total volume of chronic lesions from CAS1/4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice trended to be smaller compared to chronic lesions from CAS1/4\u003csup\u003e+/+\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, D, E). Although there was no difference in the amount of CD45\u003csup\u003e+\u003c/sup\u003e cells, CAS1/4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice had significantly lower amounts of cleaved IL-1β and percent of CD45\u003csup\u003e+\u003c/sup\u003e cells that overlapped with cleaved IL-1β compared to CAS1/4\u003csup\u003e+/+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB-G), which is consistent with their deficiency in CAS1/4 expression. In addition, while the amount of lesional IBA1 and MBP were not different between the two groups, chronic lesions from CAS1/4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice had significantly reduced amount of E06\u003csup\u003e+\u003c/sup\u003e OxPC accumulation and loss of NFH\u003csup\u003e+\u003c/sup\u003e axons compared to chronic lesions from CAS1/4\u003csup\u003e+/+\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D, H-M). These results suggest that CAS1/4 activity contributes to endogenous lipid peroxidation and chronic neurodegeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince IL-1β signaling is dependent on IL-1R1\u003csup\u003e54\u003c/sup\u003e and can promote neuroinflammation\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, we also treated mice with PBS or anakinra, an antagonist to IL-1R1 from days 28\u0026ndash;42 following OxPC deposition in the SCWM (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Compared to chronic OxPC lesions in PBS treated mice, chronic lesions in anakinra treated mice had significantly reduced amount of E06\u003csup\u003e+\u003c/sup\u003e OxPC accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, D) but significantly more IBA1\u003csup\u003e+\u003c/sup\u003e microglia/macrophages, which overlapped with E06\u003csup\u003e+\u003c/sup\u003e OxPC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, F). While lesional MBP percentage was equivalent in both treatment groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, G), anakinra treated chronic lesions had significantly greater density of OLIG2\u003csup\u003e+\u003c/sup\u003e oligodendroglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH) and NFH\u003csup\u003e+\u003c/sup\u003e axons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI) compared to PBS treated lesions. Thus, blockade of IL-1R1 in chronic OxPC lesions reduced OxPC accumulation and partially ameliorated the severity of chronic neurodegeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFinally, to validate the relevancy of OxPC deposition and IL-1β elevation in P-MS, we analyzed CD45 and E06 immunoreactivity in the NAWM and the CAL rims from fresh-frozen tissues collected from rapid autopsies of P-MS brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eJ). Compared to CD45\u003csup\u003e+\u003c/sup\u003e cells that resembled more ramified microglia in the NAWM, CD45\u003csup\u003e+\u003c/sup\u003e cells in the rims of CAL were significantly larger and more amoeboid in morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eK-M). Importantly, CAL rims had significantly greater deposition of E06\u003csup\u003e+\u003c/sup\u003e OxPC as well as significantly greater amount of E06\u003csup\u003e+\u003c/sup\u003e OxPC that overlapped with CD45\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eN, O). Total IL-1β and IL-1β\u003csup\u003e+\u003c/sup\u003e CD45\u003csup\u003e+\u003c/sup\u003e cells were also elevated in CAL rims compared to the NAWM (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eP, Q). Thus, endogenous OxPC deposition as well as reactivity of IL-1β and OxPC associated microglia/macrophages were pathological features of CAL in P-MS.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe molecular mechanisms mediating chronic neurodegeneration and MS progression are not well understood. OxPC are neurotoxic byproducts of oxidative stress and OxPC accumulation prominently associates with all stages of MS\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Here, we report that OxPC deposition in the mouse SCWM induced a chronic focal lesion with features akin to CAL from P-MS including compartmentalized neuroinflammation, demyelination, and neurodegeneration, as well as endogenous OxPC accumulation. The severity of chronic neurodegeneration and endogenous OxPC accumulation in these CAL-like lesions were significantly increased by aging and by microglia depletion. An increase of IL-1β levels was associated with lesion chronicity and with conditions that exacerbated chronic neurodegeneration and endogenous OxPC accumulation, suggesting a link between OxPC mediated pathology and IL-1β activity. Indeed, E06\u003csup\u003e+\u003c/sup\u003e OxPC immunoreactivity as well as the loss of NFH\u003csup\u003e+\u003c/sup\u003e axons were significantly lower in CAS1/4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, which have deficiency in pro-IL-1β processing\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Conversely, IL-1R1 blockade ameliorated axon loss and endogenous OxPC accumulation in chronic lesions. These results together with the observation that IL-1β and OxPC accumulated in CAL from P-MS brains, suggested that aberrant IL-1β signaling and OxPC contributes to chronic neurodegeneration in P-MS.\u003c/p\u003e \u003cp\u003eAlthough OxPC accumulation\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20 CR21\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and inflammation associated oxidative injury\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\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 are major features of MS pathophysiology, their functional involvement in P-MS is not well understood because commonly studied experimental models of MS such as EAE or LPC induced demyelination have pathologies that involve relatively low levels of OxPC and oxidative injury\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Here, we show that a one-time deposition of POVPC, a type of OxPC upregulated in MS lesions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, promoted chronic unremitting compartmentalized injury in the SCWM of mice. Chronic OxPC lesions had significant neurodegeneration in the form of axonal loss, persistent demyelination/remyelination failure, as well as accumulation of endogenous OxPC and inflammatory microglia/monoMac that upregulated NADPH oxidase and IL-1β. These pathological responses are similar to CAL disease activity in P-MS, which also involves axon loss\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, ongoing demyelination\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, microglia/monoMac reactivity\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, and NADPH oxidase upregulation\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Moreover, we found significant accumulation of OxPC and IL-1β with reactive phagocytes in CAL from P-MS brains. These observations indicate that OxPC mediated chronic lesions recapitulated key aspects of P-MS pathophysiology. Notably, the exacerbation of OxPC mediated chronic neurodegeneration in middle-aged mice is evidence that aging associated defects in OxPC mitigation contributes to the acceleration of chronic neurodegeneration as people with P-MS become older. Thus, this model will be useful for uncovering new mechanisms that regulate chronic neurodegeneration in P-MS and aging.\u003c/p\u003e \u003cp\u003eThe concentration of OxPC deposited in active lesions or CAL from MS have not been measured systematically and only one study estimated there are approximately 0.6 \u0026micro;g of OxPC per mg of protein isolated from MS brain homogenate\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. In this study, chronic lesions developed after the deposition of 5 \u0026micro;g of POVPC, which is likely within the range of OxPC concentrations potentially found in MS lesion microenvironments. Using the OxPC specific and neutralizing E06 antibody\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, we previously found minimal OxPC in the focal SCWM lesion at 1 day post POVPC injection\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, which indicates that most if not all of the injected POVPC quickly reacts with the tissue. Thus, the detection of E06\u003csup\u003e+\u003c/sup\u003e OxPC within focal OxPC lesions after 42 and 100 days reflects significant endogenous OxPC production, potentially due to similar mechanisms found in MS lesions. Notably, OxPC accumulation was minimal in chronic LPC or EAE lesions, highlighting that the pathology in these models may be different from that of chronic OxPC lesions and OxPC\u003csup\u003e+\u003c/sup\u003e CAL from P-MS.\u003c/p\u003e \u003cp\u003eThe cause of the endogenous OxPC accumulation in MS lesions remains relatively unknown. Reactive microglia/monoMac are major populations of immune cells found in CAL of P-MS and they express NADPH oxidase\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Thus, they have the capacity to produce ROS, which can promote nonenzymatic oxidation of PC into OxPC\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e in the lesion microenvironment. Similarly, microglia and monoMac with elevated NADPH oxidase expression were also the predominant immune cells in chronic OxPC lesions, suggesting they may contribute to lesion chronicity by producing ROS which leads to lipid peroxidation and endogenous OxPC formation, which in turn generate a feed-forward loop to generate more ROS and OxPC\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Alternatively, NADPH oxidase upregulation by microglia/monoMac in chronic lesions may not fully reflect in situ ROS/OxPC generation as PC oxidation can also be initiated enzymatically by 12/15-lipoxygenase\u003csup\u003e59, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e, which is also upregulated in MS\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Both of these possibilities may be investigated by generating transgenic mice with microglia/monoMac deficient in NADPH oxidase\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e or 12/15-lipoxygenase\u003csup\u003e63\u003c/sup\u003e expression. Nevertheless, since IL-1β was significantly elevated in chronic OxPC lesions and in CAL from P-MS, it may be involved in sustaining microglia/monoMac reactivity, oxidative stress, and lipid peroxidation in the lesion microenvironment. In support of this hypothesis, we previously showed IL-1β deposition in the SCWM directly promotes acute neuroinflammation and endogenous OxPC formation\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e whereas here we found blocking IL-1R1 signaling reduced OxPC mediated chronic neurodegeneration. These findings, together with recent evidence showing that OxPC can also enhance IL-1β production in both primed myeloid cells\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e and mouse peritoneal macrophages\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e suggest a potential positive feedback loop between OxPC and IL-1R1 mediated inflammatory responses. Thus, strategies to inhibit IL-1β signaling in P-MS may help to interrupt the vicious cycle of OxPC generation, neuroinflammation, and neurodegeneration. It is important to note that OxPL are abundantly generated whenever cells undergo apoptosis cell death, thus further contributing to this positive feed-forward destructive cycle\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCAL associate with chronic neurodegeneration and disability progression in people with P-MS\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. They are classified by a relatively inactive lesion core surrounded by a slowly-expanding lesion rim with ongoing myelin loss and accumulation of lipid-laden microglia/monoMac\u003csup\u003e1, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The relative composition of microglia versus monoMac in CAL remains unclear and is difficult to determine as the expression of microglia specific markers such as TMEM119 or P2RY12 becomes less stable in reactive microglia\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. While microglia and monoMac in CAL are largely characterized as proinflammatory cells\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, their functional contribution to chronic neurodegeneration remains uncertain. By fate mapping tdTomato\u003csup\u003e+\u003c/sup\u003e microglia in chronic OxPC lesions, we found that unlike in acute lesions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, tdTomato\u003csup\u003e\u0026minus;\u003c/sup\u003e monoMac re-emerged as a significant immune cell population in chronic OxPC lesion. Importantly, the amount of monoMac versus microglia was significantly increased within chronic lesions of middle-aged mice compared to lesions from young mice, which is consistent with recent findings showing that chronic injury and aging drive the accumulation of disease associated monoMac in the CNS\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Importantly, we found that middle-aged mice with greater monoMac accumulation and mice with microglia depletion had greater OxPC and IL-1β accumulation as well as worse chronic neurodegeneration compared to young mice and mice without microglia depletion, respectively. These findings together with studies showing that microglia mediated phagocytic processing of harmful lipids becomes dysregulated by aging\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR71 CR72\" citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, implicate that microglial loss and/or their dysregulation caused by chronic disease activity and aging can facilitate the acceleration of chronic neurodegeneration in P-MS.\u003c/p\u003e \u003cp\u003eIn summary, we report that OxPC deposition in the CNS induces pathology similar to CAL from P-MS including endogenous lipid peroxidation, unremitting neuroinflammation, and chronic neurodegeneration. Aging and microglial dysregulation in part through inflammatory IL-1β signaling exacerbated chronic neurodegeneration. Promoting OxPC neutralization and clearance, as for example by targeting with the E06 antibody, and/or promoting homeostatic microglia repopulation may be effective approaches to slow and/or halt chronic neurodegeneration in P-MS.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eONLINE METHODS\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eMS specimens\u003c/h2\u003e \u003cp\u003ePost-mortem human brain tissues were obtained from seven patients diagnosed with clinical and neuropathological P-MS according to the revised 2010 McDonald\u0026rsquo;s criteria\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, with full ethical approval (BH07.001, Nagano 20.332 - YP) and informed consent as approved by the CRCHUM and University of Montreal research ethics committee. As previously described\u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e, autopsy samples were cryopreserved, and lesions were classified using Luxol fast blue and H\u0026amp;E staining.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMice\u003c/h2\u003e \u003cp\u003e All experiments were conducted with ethics approval (protocol number 20220103) from the Animal Care Committee at the University of Saskatchewan under regulations of the Canadian Council of Animal Care. Female 6wk and 52wk old C57Bl/6J mice were acquired from Jackson Laboratories for in vivo experiments. CX3CR1\u003csup\u003ecreER\u003c/sup\u003e (strain 021160) mice, Ai9\u003csup\u003eTdTom\u003c/sup\u003e mice (strain 007909), and Rosa26\u003csup\u003eiDTR\u003c/sup\u003e mice (strain 007900) from The Jackson Laboratory were bred in the Lab Animal Services Unit at the University of Saskatchewan to produce male and female CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice and CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Rosa26\u003csup\u003eiDTR\u003c/sup\u003e mice for microglial fate mapping and depletion studies. Female 6-10wk old C57BL/6NJ (CAS1/4\u003csup\u003e+/+\u003c/sup\u003e, strain 005304) and B6N.129S2-Casp1tm1Flv/J (CAS1/4\u003csup\u003e/\u0026minus;\u003c/sup\u003e strain 016621) mice from Jackson Laboratories were used for IL-1β studies. Mice were maintained on a regular diet in low humidity environment on a 12-hr light/dark cycle at 21 to 23 degrees Celsius with unlimited access to food and water. Mice and littermates were randomly assigned to different experimental groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eFate mapping microglia/monoMac\u003c/h2\u003e \u003cp\u003eSix-week-old and 52-week-old CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Ai9\u003csup\u003etdTom\u003c/sup\u003e mice were intraperitoneally injected with 2 mg of tamoxifen (20 mg/ml; T5648, Sigma) dissolved in corn oil (C8267) once a day for 3 consecutive days to induce tdTomato expression in all CX3CR1\u003csup\u003e+\u003c/sup\u003e mononuclear phagocytes. Mice were then used 4 weeks after tamoxifen injection which is when tdTomato expression only labels long lived CNS resident microglia and macrophages but not monoMac derived from infiltrating monocytes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMicroglia depletion\u003c/h2\u003e \u003cp\u003eNewly weaned CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Rosa26\u003csup\u003eiDTR\u003c/sup\u003e mice were injected intraperitoneally with 2 mg tamoxifen (20 mg/ml; T5648, Sigma) dissolved in corn oil (C8267) once a day for 5 consecutive days to induce DTR expression on microglia and were used for experiments 3 weeks after tamoxifen injection. For microglia depletion during acute lesion phase, tamoxifen treated CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Rosa26\u003csup\u003eiDTR\u003c/sup\u003e mice were injected intraperitoneally with PBS or 1 \u0026micro;g of DT every other day from days 0 to 7 after OxPC deposition in the SCWM. For microglia depletion during chronic lesion phase, tamoxifen treated CX3CR1\u003csup\u003eCreER\u003c/sup\u003e:Rosa26\u003csup\u003eiDTR\u003c/sup\u003e mice were injected intraperitoneally with PBS or 1 \u0026micro;g of DT every other day from days 35 to 42 after OxPC deposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAnakinra blockade of IL-1R1\u003c/h2\u003e \u003cp\u003eSix-week-old C57Bl/6J mice were intraperitoneally injected daily with 100 \u0026micro;l of PBS or PBS containing 1 mg/kg anakinra (HY-108841, MedChemExpress) from days 28\u0026ndash;42 following OxPC deposition in the SCWM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSpinal cord surgery\u003c/h2\u003e \u003cp\u003eThe surgical procedure for OxPC spinal cord injection was performed as described from previous studies\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Briefly, mice were anesthetized with ketamine and xylazine and injected stereotactically with 0.5 \u0026micro;l PBS containing 10 mg/ml POVPC (Avanti Polar Lipids, 870606P) into the ventrolateral SCWM between the T3 and T4 vertebra. After the injection, the needle was left in place for 2 min to prevent back flow, and then the mouse was sutured and placed in a thermally controlled environment for recovery. Alternatively, equivalent amount of LPC (Sigma, L1381) was injected in the SCMW using the same approach.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEAE and tissue isolation\u003c/h2\u003e \u003cp\u003eEight- to 10-week-old female C57Bl/6J mice were subcutaneously inoculated with 50 \u0026micro;g of MOG 35\u0026ndash;55 peptide (Protein and Nucleic Acid Facility, Stanford University School of Medicine) in 100 \u0026micro;l of complete Freund\u0026rsquo;s adjuvant supplemented with 4 mg/ml heat-inactivated Mycobacterium tuberculosis H37Ra (Sigma-Aldrich), in which 50 \u0026micro;l emulsion was deposited on each side of the tail base. Intraperitoneal injection of pertussis toxin (300 ng per 200 \u0026micro;l; 180, List Biological Laboratories) was performed days 0 and 2 after MOG immunization. Mice were monitored and scored daily on a scale of 0\u0026ndash;15. EAE mice during chronic disease (day 45) were euthanized with intraperitoneal injections of ketamine and xylazine. Fifteen ml of PBS was then perfused via cardiac puncture, and the cerebellum and spinal cord were collected. The cerebellum was frozen in Optimal cutting temperature polymer (Leica), whereas the spinal cord was processed as stated above for the spinal cord injections. Cerebellar sections (sagittal) and the spinal cord sections (longitudinal) were cut into 20-\u0026micro;m sections using a cryostat and collected onto Superfrost Plus microscope slides (VWR) and stored at \u0026minus;\u0026thinsp;20\u0026deg;C before analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSpinal cord tissue isolation for histology and microscopy analysis\u003c/h2\u003e \u003cp\u003eMice were euthanized with intraperitoneal ketamine and xylazine overdose after 7-, 42-, or 100-days post-surgery. Ten ml of PBS followed by 10 ml of 4% paraformaldehyde in PBS were perfused via cardiac puncture. The spinal cord was then dissected from the back of the mouse, and the tissue containing the T3-T4 inject site was collected into 4% paraformaldehyde in PBS for fixation overnight at 4 degrees. Thereafter, spinal cords were transferred to 30% sucrose solution for dehydration for at least 48h and frozen in FSC 22 Frozen Section Media (Leica). With a cryostat (ThermoFisher Scientific), spinal cord tissue was cut into 20 \u0026micro;m coronal sections and collected on to Superfrost Plus microscope slides (VWR). Tissues were stored at -20 degrees prior to staining and analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eThe following primary antibodies were used for immunofluorescence microscopy: mouse IgM E06 anti-OxPC (5 \u0026micro;g/ml, generously provided by Witztum and Tsimikas labs), rabbit anti-human/mouse IBA1 (1:1000, Wako 019-19741), chicken anti-human/mouse IBA1 (1:1000, Synaptic Systems 234 009), rat anti-mouse MBP (1:200, Abcam ab7349), rat anti-mouse CD11b (1:200, ThermoFisther 14-0112-82), goat anti-human/mouse OLIG2 (1:200, R\u0026amp;D Systems AF2418), rabbit anti-mouse NFH (1:1000, Encor Biotechnology RPCA-NF-H), chicken anti-mouse NFH (1:1000, Encor Biotechnology CPCA-NF-H), rat anti-mouse iNOS (1:100, ThermoFisher 14-5920-82), mouse anti-human/mouse IL-1β (1:100, Cell Signaling 12242S), rat anti-human/mouse CD45 (1:200, ThermoFisher MA5-17687), rabbit anti-mouse cleaved IL-1β (1:100 Cell Signaling 63124S), Rabbit anti-mouse Arg1 (1:200, Cell Signaling 93668S, rat anti-mouse CD16/32 (1:100, BD Pharmingen 553141). rabbit anti-mouse P22phox (1:200 Cell Signaling 37570S), chicken anti-human/mouse GFAP (1:1000, Biolegend 829401), rabbit anti-human/mouse βAPP (1:200, Thermo Fisher Scientific 36-6900), rabbit anti-human/mouse Ki67 (1:200, Abcam ab15580), and rabbit anti-mouse p16\u003csup\u003eINK\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003eA\u003c/sup\u003e (1:100, Cell Signaling 29271S).\u003c/p\u003e \u003cp\u003eThe following secondary antibodies from Jackson ImmunoResearch were used at 1:400 dilution: Alexa Fluor 488 donkey anti-mouse IgM, Alexa Fluor 488 donkey anti-mouse IgG, Cyanine Cy3 donkey anti-chicken IgY, Alexa Fluor 647 donkey anti-rat IgG, Cyanine Cy3 donkey anti-rat IgG, Alexa Fluor 488 donkey anti-goat IgG, Alexa Fluor 647 donkey anti-rabbit IgG.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMouse spinal cord histology\u003c/h2\u003e \u003cp\u003eFor eriochrome cyanine (EC) and neutral red (NR) visualization of serial spinal cord lesions, spinal cord sections were stained as previously described\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Alternatively, serial spinal cord lesions were also visualized by immunofluorescence labeling of MBP. Brightfield images were then acquired using the Olympus VS110 Slidescanner with a 10x 0.4 NA air objective. Lesion ROIs were drawn based on demyelinated areas in the SCWM that have lower EC staining and higher NR staining and their total areas were quantified using the CEllSens Dimension software (Olympus). Lesion volume was estimated by multiplying the distance separating each serial section (400 \u0026micro;m) by the sum of lesion areas in the serial spinal cord sections from each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLabeling tissues for immunofluorescence confocal microscopy\u003c/h2\u003e \u003cp\u003eAs previously described\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, slides with mouse spinal cord samples were warmed to room temperature (RT) for 10 min. When MBP staining is required, slides were delipidated by successive wash of 50%, 70%, 90%, 95%, 100%, 95%, 90%, 70%, and 50% ethanol. Then, samples were rehydrated in PBS for 10 min and permeabilized with 0.2% Triton-X100 in PBS for 10 min. Samples were then blocked with donkey blocking solution (PBS, 10% donkey serum, 1% BSA, 0.1% cold fish stain gelation, 0.1% Triton X-100, 0.05% Tween-20) for 1h at RT or overnight at 4 degrees. Alternatively, E06 antibody staining, 5 \u0026micro;g/ml of purified Rat Anti-Mouse CD16/CD32 Fc blocking antibody (5 \u0026micro;g/ml, BD Pharmingen) was added to the blocking buffer. After blocking, samples were incubated with primary antibodies in antibody dilution buffer (PBS, 1% BSA, 0.1% cold fish stain gelation, 0.1% Triton X-100) for overnight incubation at 4 degrees. Samples were then washed 3 times, 5 min each with PBS and 0.2% Tween-20 and incubated with secondary antibodies and 1 \u0026micro;g/ml of DAPI resuspended in the antibody dilution buffer for 1h at RT. For samples with high potential of autofluorescence, slides were also blocked using the TrueBlack Lipofuscin Autofluorescence Quencher (Biotium) in accordance with manufacturer\u0026rsquo;s instructions. Finally, slides were washed 3 times using PBS with 0.2% Tween-20, 5 min each, and coverslips were mounted onto the slides using Fluoromount-G solution (SouthernBiotech).\u003c/p\u003e \u003cp\u003eFor post-mortem MS tissue samples, after slides were warmed to RT, they were fixed with 4% paraformaldehyde for 10 min, then washed in PBS for 10 min to remove excess PFA. The remaining steps were the same as the mouse spinal cord samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence microscopy\u003c/h2\u003e \u003cp\u003eImmunofluorescence images were acquired using the Leica TCS Sp8 laser confocal microscope at RT, using the 10x 0.40 NA air object or the 25x 0.5 NA water objective. The 405 nm, 488 nm, 552 nm, and 640 nm lasers were used to excite the fluorophores from antibodies bound to samples and detected by two low dark current Hamamatsu PMT detectors and two high sensitivity hybrid detectors. Images were acquired in 8-bits, in a z-stack using unidirectional scanning, 1 airy unit pinhole, 0.75x zoom, and 0.57 \u0026micro;m optical sections and 2048 x 2048 pixels xy resolution. Alternatively, images were acquired using the Zeiss LSM700 confocal microscope with a 20x 0.8 NA air objective using similar settings. Images were also acquired using the Zeiss Axio Observer 7 widefield microscope with Colibri 7 LED illumination and a 25x 0.85 NA water objective, followed by constrained iterative deconvolution processing. Equal laser, gain, offset, and exposure settings to maximize contrast and minimize saturation were consistently used for all samples within experiment sets. A sample slide stained with only the secondary antibodies and DAPI was used for each experiment to control for non-specific secondary immunofluorescence. Leica Application Suite X or Zeiss Zen Black software was used for image acquisition, ImageJ was used for image threshold and particle analysis.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eImage analysis\u003c/h2\u003e \u003cp\u003eZ-stack confocal images of spinal cords were analyzed with ImageJ (Fiji, NIH) as previously described\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Briefly, maximum intensity projections were created for each channel/marker z-stack and converted from 8-bit to RGB. The lesion ROI or equivalent area in the contralateral normal appear white matter (NAWM) was drawn while the area outside the ROI was not analyzed. Positive signal was determined using the color brightness threshold set consistently using a predetermined value by comparing the secondary antibody-stained control and NAWM. Lesion ROIs were drawn based on markers that define the SCWM lesion area such as CD16/32, IBA1, or MBP. The analyze particles function was then used to quantify the positive signals in each ROI. To avoid bias, the same threshold values for setting the positive signal, as well as the size and circularity settings for particle analysis were used for all samples in each experimental set. For representative images shown, maximum intensity projection of each channel/marker in a z-stack were merged and displayed using pseudo colors. Only brightness and contrast settings were adjusted in ImageJ, and consistently between samples for better displaying the images.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eStatistics and reproducibility\u003c/h2\u003e \u003cp\u003eData were collated in Microsoft Excel and graphs were generated using GraphPad Prism 10.1 (LaJolla, CA). Data shown are the individual data points where each point on a graph represents a separate mouse. The mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD are also shown. No sample size calculation was performed. Sample size was determined based on previously published results\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e and based the cost of experiment, feasibility of the experiment, as well as the availability of sex and aged matched mice. No data was excluded from the analyses. Sample sizes are reported in the figure legends and only one measurement is recorded per sample. Littermate mice were randomly selected for each experimental condition and treatment. Blinding was not conducted. For analysis of statistical significance between the means of two or more treatment groups against the control group, one-way ANOVA with Tukey\u0026rsquo;s multiple comparison test was used. Two-tailed, unpaired t-test was used to compared data with only two groups. Kolmogorov-Smirnov test was used to verify the normal distribution of data. Specific P-values are reported in each figure where results are considered statistically significant where p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll data are available upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.D. acknowledges operating grant support from the University of Saskatchewan College of Medicine, the Natural Sciences and Engineering Research Council of Canada, MS Canada, Saskatchewan Health Research Foundation, and Brain Canada Foundation. A.S. and J.P. acknowledge undergraduate Biomedical Project research support from the College of Medicine, University of Saskatchewan. J.P. and K.T. also acknowledge undergraduate student research award support from the Natural Sciences and Engineering Research Council of Canada.\u0026nbsp;A.P. holds a Tier 1 senior Canada Research Chair in Multiple Sclerosis and is funded by the CIHR, MS Canada, the International Progressive MS Alliance, the Canadian Foundation for Innovation and the National MS Society (USA).\u0026nbsp;We also thank Histology and Cancer Cluster Core Facilities at the University of Saskatchewan, as well as the Hotchkiss Brain Institute Advanced Microscopy Platform Facility at the University of Calgary, for technical help.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.D. conceived the project and designed experiments. R.Y., B.M.L., Y.D., A.S., and K.T performed experiments and analyzed data. D.K.K. provided support and data for EAE experiments. S.Z., W.K., S.L, and A.P. provided P-MS brain specimens and their characterization. S.T. and J.L.W. provided critical reagent support. Y.D. supervised the overall study and wrote the manuscript. All authors reviewed and edited the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYong, H.Y.F. \u0026amp; Yong, V.W. Mechanism-based criteria to improve therapeutic outcomes in progressive multiple sclerosis. Nat Rev Neurol 18, 40\u0026ndash;55 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUniversity of California, S.F.M.S.E.T., \u003cem\u003eet al.\u003c/em\u003e Silent progression in disease activity-free relapsing multiple sclerosis. 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STAR Protoc 2, 100853 (2021).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4792293/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4792293/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxidized phosphatidylcholines (OxPC) are neurotoxic byproducts of oxidative stress elevated in the central nervous system (CNS) during progressive multiple sclerosis (P-MS). How OxPC contribute to the pathophysiology of P-MS is unclear. Here, we report that OxPC deposition in the CNS of mice induces a chronic compartmentalized lesion with pathological features similar to chronic active lesions found in P-MS. Using this new model, we found that while microglia protected the CNS from chronic neurodegeneration, they were also replaced by monocyte derived macrophages in chronic OxPC lesions. Aging, a risk factor for P-MS, altered microglial composition and exacerbated neurodegeneration in chronic OxPC lesions. Amelioration of disease pathology in caspase 1/4 deficient mice and by blockade of IL-1R1 indicate IL-1β signaling contributes to chronic OxPC accumulation and neurodegeneration. These results highlight OxPC and IL-1β as potential drivers of chronic neurodegeneration in MS and suggest that their neutralization may be effective for treating P-MS.\u003c/p\u003e","manuscriptTitle":"Oxidized phosphatidylcholines induce chronic neurodegeneration partly through IL-1β mediated positive feedback","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-05 00:24:50","doi":"10.21203/rs.3.rs-4792293/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-neuroscience","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"neuro","sideBox":"Learn more about [Nature Neuroscience](http://www.nature.com/neuro/)","snPcode":"","submissionUrl":"","title":"Nature Neuroscience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"67564300-a2c4-4653-ae6d-473a09bf4d6a","owner":[],"postedDate":"August 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35459290,"name":"Biological sciences/Neuroscience/Neuroimmunology"},{"id":35459291,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Multiple sclerosis"}],"tags":[],"updatedAt":"2025-12-02T08:07:08+00:00","versionOfRecord":{"articleIdentity":"rs-4792293","link":"https://doi.org/10.1038/s41593-025-02113-y","journal":{"identity":"nature-neuroscience","isVorOnly":false,"title":"Nature Neuroscience"},"publishedOn":"2025-12-01 05:00:00","publishedOnDateReadable":"December 1st, 2025"},"versionCreatedAt":"2024-08-05 00:24:50","video":"","vorDoi":"10.1038/s41593-025-02113-y","vorDoiUrl":"https://doi.org/10.1038/s41593-025-02113-y","workflowStages":[]},"version":"v1","identity":"rs-4792293","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4792293","identity":"rs-4792293","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}