In multiple sclerosis (MS), accumulation of disability is driven by CNS-compartmentalized inflammation. This inflammatory process involves activated microglia and astrocytes, which contribute to neuroaxonal damage which in turn accelerates disease progression. Activated glial cells express 18-kDa translocator protein (TSPO), and TSPO-binding radioligands and positron emission tomography (PET) imaging can be used to quantitate glial activation in vivo. The aim of this study was to evaluate the longitudinal evolution of glial activation in untreated cohorts of relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients over one-year follow-up, and to explore how a change in glial activation associates with later imaging and clinical outcomes. Eighteen untreated MS patients (RRMS n = 8, SPMS n = 10) were studied. Expanded disability status scale (EDSS), brain MRI and TSPO-PET scans using [11C]PK11195 were performed at baseline and one year later. Distribution volume ratio (DVR) of [11C]PK11195-binding, and the proportion of TSPO-high voxels at baseline in the normal appearing white matter (NAWM) and other regions of interest were compared to the respective parameters in follow-up scans. Chronic lesions were phenotyped at baseline and at follow-up according to their TSPO-PET-binding patterns, and TSPO-expressing lesions were further characterized using postmortem immunopathological staining. Extended follow-up was obtained after 4-11 years with EDSS available for 18 patients and MR imaging available from 13 patients. TSPO-signal was higher among SPMS compared to RRMS patients at baseline. During one-year follow-up, TSPO uptake remained stable in RRMS patients in all regions of interest. Among the SPMS patients, the proportion of active voxels in the NAWM increased significantly over one-year follow-up. A greater proportion of lesions acquired a rim-active phenotype among SPMS compared to RRMS. According to forward-type stepwise multiple linear regression, change in the proportion of active voxels in the NAWM over one year and baseline body-mass-index were best predictors of later development of brain atrophy (R2 = 0.69). Our study provides novel information about the natural evolution of CNS-compartmentalized inflammation and demonstrates an important link between NAWM TSPO-signal and later adverse outcomes among MS patients, supporting the notion that diffuse glial activation in the NAWM contributes to disease progression.

Objective To investigate which baseline clinical and imaging characteristics best predict TSPO-PET-measurable reduction in glial activation following treatment of multiple sclerosis (MS), to utilize this information for designing more efficient biomarker-based clinical trials targeting glial activation.Methods This study pooled data from 47 pwMS treated with various approved disease-modifying therapies and 18 untreated pwMS with TSPO-PET imaging before and after. Therapeutic response was quantified using [11C]PK11195 distribution volume ratio and percentage of active voxels in seven brain regions. Variables predicting therapeutic response were identified using linear mixed-effect models. Power calculation was used to estimate the required sample size for predictor-enriched cohorts.Results High baseline TSPO binding in the white matter (HOT-PET) was identified as the best predictor for reduction in glial activation following treatment in 6 of 14 (43%) PET variables. Internal validation confirmed that treated HOT-PET patients showed enhanced therapeutic response compared with non-HOT-PET patients in 9 of 14 (64%) PET variables. The percentage of active voxels in the white matter was the best PET variable at capturing a significant therapeutic effect, with a Cohen's d effect size of -0.779 (95% confidence interval -1.332; -0.207). In this cohort, enrichment for HOT-PET patients markedly reduced the sample size required to show a positive treatment effect.Interpretation HOT-PET patients are more likely to benefit from neuroinflammation-targeting treatments compared to non-HOT-PET patients. Accordingly, enriching trial cohorts for individuals with greater neuroinflammatory burden could improve statistical power and reduce the required number of participants in trials targeting harmful glial activation in MS.

Aberrant and sustained activation of microglia is implicated in the progression and severity of multiple sclerosis (MS). However, whether intrinsic alterations in microglial function impact the pathogenesis of this disease remains unclear. We conducted transcriptomic and functional analyses of microglia-like cells (iMGLs) differentiated from induced pluripotent stem cells (iPSCs) from patients with MS (pwMS) to answer this question. The pwMS showed increased innate immune cell activity via 18-kDa translocator protein positron emission tomography imaging. After confirming that the differentiated iMGLs transcriptional profile is determined by the microglial cell type, comparative studies were performed to identify the transcriptional and functional differences between iMGLs from pwMS and healthy controls. Importantly, MS iMGLs presented cell-autonomous differences in their regulation of inflammation, both in the basal state and following inflammatory lipopolysaccharide challenge. Through transcriptomic profiling, we showed that MS iMGLs display increased expression of genes upregulated in MS pathology. Furthermore, upregulated genes in MS iMGLs were associated with immune receptor activation, antigen presentation, and the complement system. MS iMGLs demonstrated transcriptional similarities to lesion-specific microglia in MS, marked by upregulation of immune-related genes and pathways, including those involved in antigen presentation. Finally, functional analyses indicated that the transcriptional changes in MS iMGLs corresponded with modulation of cytokine secretion and increased phagocytosis. Together, our results provide evidence of putative cell-autonomous microglial activation in pwMS and identify transcriptomic and functional changes that recapitulate the phenotypes observed in vivo in microglia from pwMS. These findings indicate that MS disease-specific iPSCs are valuable tools for studying disease-specific microglial activation in vitro and highlight microglia as potential therapeutic targets in MS.

Background Predicting disease progression in multiple sclerosis (MS) remains challenging. PET imaging with 18 kDa translocator protein (TSPO) radioligands can detect microglial and astrocyte activation beyond MRI-visible lesions, which has been shown to be highly predictive of disease progression. We previously demonstrated that nuclear magnetic resonance (NMR)-based metabolomics could accurately distinguish between relapsing-remitting (RRMS) and secondary progressive MS (SPMS). This study investigates whether combining TSPO imaging with metabolomics enhances predictive accuracy in a similar setting.Methods Blood samples were collected from 87 MS patients undergoing PET imaging with the TSPO-binding radioligand 11C-PK11195 in Finland. Patient disability was assessed using the expanded disability status scale (EDSS) at baseline and 1 year later. Serum metabolomics was performed to identify biomarkers associated with TSPO binding and disease progression.Results Greater TSPO availability in the normal-appearing white matter and perilesional regions correlated with higher EDSS. Serum metabolites glutamate (p=0.02), glutamine (p=0.006), and glucose (p=0.008), detected by NMR, effectively distinguished future progressors. These three metabolites alone predicted progression with the same accuracy as TSPO-PET imaging (AUC 0.78; p=0.0001), validated in an independent cohort. Combining serum metabolite data with PET imaging significantly improved predictive power, achieving an AUC of 0.98 (p<0.0001).Conclusion Measuring three specific serum metabolites is as effective as TSPO imaging in predicting MS progression. However, integrating TSPO imaging with serum metabolite analysis substantially enhances predictive accuracy. Given the simplicity and affordability of NMR analysis, this approach could lead to more personalised, accessible treatment strategies and serve as a valuable tool for clinical trial stratification.

Current multiple sclerosis (MS) treatments reduce relapse activity but have limited impact on disease progression. Clinical trials targeting progression often fail because of insufficient understanding of its underlying mechanisms. This study analyzed a clinically well-characterized MS autopsy cohort from the Netherland Brain Bank (186 individuals) from which we selected donors exhibiting opposite disease trajectories of slow versus rapid progression. We performed extensive unbiased histology and spatial transcriptomics, which unveiled a distinct MS lesion type marked by an extensive myeloid cell rim with cellular and transcriptional signatures of innate immune activation, inflammatory cytokine production, unfolded protein response and apoptosis. Presence of this particular lesion type was linked to rapid disease progression. An independent translocator protein 18-kDa positron emission tomography study (114 individuals) validates the association between lesions with a broad myeloid cell rim and disease progression in individuals with MS. Our findings offer crucial insights into the mechanisms behind MS progression, identifying broad rim lesions as a biomarker for rapid disease progression and potentially guiding patient selection for future therapeutic trials targeting central nervous system intrinsic inflammation.

BACKGROUND:Multiple sclerosis (MS) progression independent of relapses is driven by brain innate immune cell activation. The aim of this study was to evaluate the association between chitinase-3-like protein 1 (CHI3L1), expressed in brain by astrocytes and microglia, measured from blood and smouldering inflammation measured using 18 kDa translocator protein (TSPO) positron emission tomography (PET) in patients with MS. METHODS:The study cohort included 55 patients with MS (25 progressive MS (PMS) and 30 relapsing remitting MS (RRMS)) and 17 healthy controls (HC). CHI3L1 was measured with commercial ELISA from plasma samples. A subcohort (44 MS and 9 HC) underwent TSPO-PET to assess [11C]PK11195 distribution volume ratio (DVR) and MRI concurrent to blood sampling. These imaging outcomes were used in respective correlation and linear regression analyses. RESULTS:CHI3L1 concentration in plasma was higher in PMS (23.5 ng/mL) compared with HC (16.8 ng/mL, p=0.0055) and RRMS (19.3 ng/mL, p=0.049). CHI3L1 associated with brain [11C]PK11195 DVR in all MS (standardised estimate 0.89, 95% CI 0.23 to 1.55, p=0.010) and in PMS (Spearman correlation ρ=0.58, 95% CI 0.058 to 0.86, p=0.032). Additionally, CHI3L1 was associated with smaller brain volume in both MS (-0.75, -1.38 to -0.11, p=0.023) and PMS (ρ=-0.56, -0.83 to -0.095, p=0.021). Furthermore, CHI3L1 was associated with Expanded Disability Status Scale (0.70, 0.12 to 1.28, p=0.019) and age (0.93, 0.37 to 1.48, p=0.002) among all patients with MS. CONCLUSIONS:Association of CHI3L1 with glial activation and brain volume loss identifies plasma CHI3L1 as a promising biomarker for smouldering inflammation and MS progression-related pathology.

Background:Multiple sclerosis (MS) manifests clinically as relapsing disease (relapsing-remitting MS (RRMS)), progressive disease, or as combination of these phenotypes. The underlying pathology for relapses and focal inflammatory activity is driven by adaptive immune cells, whereas brain-compartmentalized pathology promoted by innate immune cell activation likely contributes to progression. Objectives:To explore the usability of various imaging and soluble biomarkers in predicting change in clinical phenotype from RRMS to secondary progressive MS (SPMS). Design:Prospective longitudinal study. Methods:Twenty-three RRMS patients aged 40-50 years had clinical evaluation, brain MR imaging, serum neurofilament light and glial fibrillary acidic protein (GFAP) measurements, and brain positron emission tomography with translocator protein (TSPO)-binding radioligand [11C](R)-PK11195 at baseline. Patients were followed for 5 years and assessed for signs of conversion to SPMS at the end of follow-up. Evolution to SPMS was determined based on an increased Expanded Disability Status Score and significant accrual of clinical symptoms. Results:After 5 years, 8/23 (35%) patients had converted to SPMS. At baseline, they had increased TSPO-binding in the normal appearing white matter, thalamus, and perilesional area compared to patients who did not convert to SPMS. The proportion and number of TSPO-rim-active lesions were higher among patients developing SPMS. Higher concentration of GFAP and more pronounced thalamic atrophy were also observed among the SPMS convertors. Conclusion:The results suggest that imaging and serum biomarkers reporting on compartmentalized central nervous system inflammation support identification of MS patients at risk of SPMS conversion. Evaluation of thalamic atrophy and measurement of soluble biomarkers can be implemented in the assessment of individual patients' progression risk in daily clinical practice. This can help in identifying patients who are at greatest need of smoldering pathology-targeting therapy. Larger studies are needed to validate these results. Trial registration:NCT3134716 role of microglia in the pathogenesis of progressive multiple sclerosis, https://clinicaltrials.gov/study/NCT03134716.

Background:Serum glial fibrillary acidic protein (sGFAP) is a promising biomarker for multiple sclerosis (MS) disease progression. Elevated sGFAP levels are considered to reflect ongoing astrocyte-related pathology in the central nervous system. Objectives:To study whether sGFAP levels associate with 18 kDa translocator protein (TSPO) availability in MS brain. TSPO is a mitochondrial molecule that is expressed by activated microglia and astrocytes. Design:Cross-sectional multimodal biomarker correlation study. Methods:We included 80 people with MS (66 relapsing-remitting and 14 progressive MS, 69% women), and 11 healthy control participants (73% women). sGFAP was measured using single molecule array (Simoa®) technology in combination with 3T magnetic resonance imaging and positron emission tomography (PET) using a TSPO-binding [11C]PK11195 radioligand. Results:sGFAP was higher among people with progressive MS (median 122 pg/ml) compared to healthy controls (median 59 pg/ml, p = 0.0002) or participants with relapsing-remitting MS (median 77 pg/ml, p = 0.0056). Among people with MS, higher sGFAP associated with higher volume of chronic lesions with increased TSPO activity (r = 0.36, p = 0.0011) and with thalamic TSPO activity (r = 0.30, p = 0.0069), as well as with T1 and T2 lesion loads (r = 0.38, 0.41, p = 0.0005, 0.0002, respectively). Smaller normal-appearing white matter (r = -0.36, p = 0.0009), cortical gray matter, and thalamus volumes (r = -0.39, p = 0.0003 for both) correlated with higher sGFAP. In regression analyses, the volume of TSPO-expressing lesions, together with age and MS disease-modifying treatment status, explained 27% of the variation in sGFAP. Conclusion:sGFAP associates with adverse magnetic resonance imaging and PET imaging outcomes. The association between a high prevalence of TSPO-expressing white matter lesions and high sGFAP suggests that lesion-associated glial activity promotes MS progression partially via astrocyte-driven mechanisms. A combination of various soluble biomarkers and PET ligands for specific cell types may add to the understanding of progression-promoting cellular mechanisms in the brain. Trial registration:ClinicalTrials.gov NCT03134716, NCT03368677, NCT04126772, NCT04239820, https://clinicaltrials.gov.

Background PET imaging of activated microglia has improved our understanding of the pathology behind disability progression in MS, and pro-inflammatory microglia at ‘smoldering’ lesion rims have been implicated as drivers of disability progression. The P2X7R is upregulated in the cellular membranes of activated microglia. A single-tissue dual-input model was applied to quantify P2X7R binding in the normal appearing white matter, perilesional areas and thalamus among progressive MS patients, healthy controls and newly diagnosed relapsing MS patients. Results Overall, tracer uptake in the MS brain was not significantly higher compared to HCs. In the 3 mm perilesional rim of all T1 lesions, tracer binding was higher among relapsing patients compared to progressive patients. Tracer binding was higher in males compared to females. Disease duration correlated with tracer binding in the normal appearing white matter. Age correlated negatively with tracer binding in the perilesional rims. Conclusions Binding estimates obtained with the dual-input model were consistent with the expected distribution of P2X7Rs in the MS brain. According to our study, [11C]SMW139-binding may capture a glial cell phenotype significant in early development of chronic active lesions in relapsing stages of MS. Only tentative evidence for the applicability of [11C]SMW139 to detect MS-related diffuse smoldering inflammation was obtained.

BackgroundMales with multiple sclerosis (MS) have a higher risk for disability progression than females, but the reasons for this are unclear.ObjectiveWe hypothesized that potential differences in TSPO-expressing microglia between female and male MS patients could contribute to sex differences in clinical disease progression.MethodsThe study cohort consisted of 102 MS patients (mean (SD) age 45.3 (9.7) years, median (IQR) disease duration 12.1 (7.0–17.2) years, 72% females, 74% relapsing–remitting MS) and 76 age- and sex-matched healthy controls. TSPO-expressing microglia were measured using the TSPO-binding radioligand [11C](R)-PK11195 and brain positron emission tomography (PET). TSPO-binding was quantified as distribution volume ratio (DVR) in normal-appearing white matter (NAWM), thalamus, whole brain and cortical gray matter (cGM).ResultsMale MS patients had higher DVRs compared to female patients in the whole brain [1.22 (0.04) vs. 1.20 (0.02), p = 0.002], NAWM [1.24 (0.06) vs. 1.21 (0.05), p = 0.006], thalamus [1.37 (0.08) vs. 1.32 (0.02), p = 0.008] and cGM [1.25 (0.04) vs. 1.23 (0.04), p = 0.028]. Similarly, healthy men had higher DVRs compared to healthy women except for cGM. Of the studied subgroups, secondary progressive male MS patients had the highest DVRs in all regions, while female controls had the lowest DVRs.ConclusionWe observed higher TSPO-binding in males compared to females among people with MS and in healthy individuals. This sex-driven inherent variability in TSPO-expressing microglia may predispose male MS patients to greater likelihood of disease progression.

BACKGROUND AND OBJECTIVES:Multiple sclerosis (MS) is a chronic inflammatory disease coupled with neurodegenerative processes affecting both the white matter and gray matter (GM) in the CNS. Several histopathologic studies have reported a reduction in synaptic density in various areas of the brain. However, this pathologic feature is yet an unexplored entity among people with MS (pwMS). Therefore, we sought to investigate synaptic loss in vivo by quantifying the synaptic vesicle glycoprotein 2A using [11C]UCB-J-PET imaging and to explore associations with clinical and cognitive measures. METHODS:Ten pwMS and 8 healthy controls (HCs) underwent high-resolution [11C]UCB-J-PET imaging and MRI. SV2A availability was determined using the tissue-to-plasma concentration ratio at equilibrium (distribution volume; VT). We furthermore explored associations between PET imaging results and clinical and cognitive measures in pwMS (assessed with the Expanded Disability Status Scale (EDSS) and Symbol Digit Modalities Test [SDMT]). In addition, we considered volumetric, clinical, and cognitive measures during a 5-year period before PET imaging. RESULTS:Ten pwMS (7 women [70%], median [interquartile range] age, 53 [50-56]) years were compared with 8 HCs (6 women [75%]; age, 51 [50-70] years). PwMS had a significantly lower SV2A availability in the cortical GM (pwMS: mean [SD], VT = 15.65 mL/cm3 [2.26]; HCs: VT = 18.14 mL/cm3 [2.09]; p = 0.029, t test), as well as in several subcortical regions. Moreover, a lower SV2A availability in cortical GM correlated significantly with reduced SDMT values in pwMS (r = 0.071, p = 0.021; Spearman correlation coefficient). No association between physical disability (measured using EDSS) and SV2A availability was found. DISCUSSION:Using in vivo [11C]UCB-J PET imaging, we provide evidence of reduced synaptic density in pwMS. Furthermore, the results reveal a link between synaptic loss and cognitive impairment. These findings highlight the potential of [11C]UCB-J PET imaging as a promising tool for assessing clinically relevant aspects of GM pathology in MS.

BACKGROUND AND PURPOSE:The aim was to study brain innate immune cell activation in teriflunomide-treated patients with relapsing-remitting multiple sclerosis.METHODS:Imaging with 18-kDa translocator protein positron emission tomography (TSPO-PET) using the [11 C]PK11195 radioligand was employed to assess microglial activity in the white matter, thalamus and areas surrounding chronic white matter lesions in 12 patients with relapsing-remitting multiple sclerosis who had been treated with teriflunomide for at least 6 months before inclusion. Magnetic resonance imaging (MRI) was used to measure lesion load and brain volume, and quantitative susceptibility mapping (QSM) was used to detect iron rim lesions. These evaluations were repeated after 1 year of inclusion. Twelve age- and gender-matched healthy control subjects were imaged for comparison.RESULTS:Half of the patients had iron rim lesions. In TSPO-PET, the proportion of active voxels indicating innate immune cell activation was slightly greater amongst patients compared with healthy individuals (7.7% vs. 5.4%, p = 0.033). The mean distribution volume ratio of [11 C]PK11195 was not significantly different in the normal-appearing white matter or thalamus amongst patients versus controls. Amongst the treated patients, no significant alteration was observed in positron emission tomography distribution volume ratio, the proportion of active voxels, the number of iron-rim-positive lesions, lesion load or brain volume during follow-up.CONCLUSIONS:Compared to controls, treated patients exhibited modest signs of diffuse innate immune cell activity, which was unaltered during follow-up. Lesion-associated smoldering inflammation was negligible at both timepoints. To our knowledge, this is the first study applying both TSPO-PET and QSM-MRI to longitudinally evaluate smoldering inflammation.

Objective: To evaluate how natalizumab treatment impacts innate immune cell activation at chronic lesion edge. Background: Increased innate immune cell activation has been associated with multiple sclerosis progression and neurodegeneration. We have demonstrated a reduction in microglial activation in the normal appearing white matter (NAWM) after natalizumab treatment. Later, a TSPO-PET-based method for phenotyping of chronic T1-lesions based on microglial activation in lesion core and at rim was developed. The impact of high-efficacy MS-treatments on microglial activation at the edge of chronic active lesions is not yet known. Design/Methods: 10 MS-patients [age 49.15 (±9.33) years] underwent PET-imaging using 11C-PK11195 radioligand and conventional MR-imaging before and after 1-year treatment with natalizumab. For comparison, 10 MS-patients [age 47.32 (±10.63)] with no disease modifying therapy were scanned similarly. T1-hypointense lesions were identified from MR-images at both time points and lesions were phenotyped based on the proportion of highly active PET-voxels at lesion rim and in lesion core. Disability assessment using Expanded Disability Status Scale was performed at time of imaging. Results: At baseline, natalizumab-treated patients had in total 24 rim-active (15% of all chronic lesions), 81 overall-active (50%) and 58 inactive (36%) lesions. The untreated cohort had 36 (15%) rim-active, 140 (57%) overall-active and 71 (29%) inactive lesions. The proportions of lesion types were unaltered during one-year follow-up in both groups, but the proportion of active voxels at the chronic active lesion rim was reduced more among natalizumab-treated patients compared to the untreated cohort [median change −7.7% (IQR −11.2– −4.5) vs. −0.5% (−2.4–0.7), p=0.015, Wilcoxon]. Similar changes were observed in the NAWM. Conclusions: TSPO-PET-imaging enables longitudinal evaluation of smoldering inflammation associated with chronic MS lesions. One-year treatment with natalizumab reduced the inflammatory burden at the active rim of chronic MS lesions but did not impact the proportions of the chronic lesion subtypes. Disclosure: Ms. Nylund has received research support from Finnish MS Foundation. Ms. Nylund has received research support from Maire Taponen Foundation. Ms. Nylund has received research support from Finnish Cultural Foundation. Markus Matilainen has nothing to disclose. Dr. Sucksdorff has nothing to disclose. Mr. Polvinen has nothing to disclose. Dr. Airas has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Genzyme. Dr. Airas has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Novartis. Dr. Airas has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Merck. Dr. Airas has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Roche.