PET Imaging of Synaptic Density Loss in Patients with Multiple Sclerosis

PET Imaging of Synaptic Density Loss in Patients with Multiple Sclerosis | 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 PET Imaging of Synaptic Density Loss in Patients with Multiple Sclerosis Chao Zheng, Pou Hong Justin Chia, Takuya Toyonaga, Junchao Tong, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6769442/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Synaptic loss is increasingly recognized as a key pathological feature in multiple sclerosis (MS), contributing to disease progression and cognitive dysfunction. Synaptic vesicle glycoprotein 2A (SV2A) positron emission tomography (PET) imaging has emerged as a promising tool for non-invasively quantifying synaptic density in vivo. Here, we evaluated the potential of SV2A PET imaging in an experimental autoimmune encephalomyelitis (EAE) mouse model and translated the findings to MS patients. In EAE mice, dynamic [18F]SynVesT-1 PET imaging revealed a significant global reduction in tracer uptake, with a nearly 30% decrease in regional distribution volume (VT) across all analyzed brain regions (p < 0.0001). Correspondingly, in vitro autoradiography on rodent EAE brains corroborated the preclinical PET imaging results. We also investigated potential synaptic loss in the spinal cord of EAE mice and observed a significant decrease in SV2A levels in both the cervical and lumbar sections. In a clinical PET research study, [11C]UCB-J was used to image in MS patients (n=5) and age-matched healthy controls (n=6), and MS patients revealed a 16.6% reduction in cortical SV2A binding (p = 0.043), with significant regional reductions in the caudate (25.6%, p = 0.026), hippocampus (18.5%, p = 0.04), and frontal cortex (16.3%, p = 0.048). These findings demonstrate the application of SV2A PET imaging as a sensitive and quantitative biomarker of synaptic pathology in MS. The consistent reductions in SV2A binding observed in both preclinical and clinical research highlight the role of synaptic degeneration in MS and underscore the utility of SV2A PET imaging in MS research. Biological sciences/Biotechnology Health sciences/Biomarkers/Diagnostic markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Multiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS), characterized by demyelination, gliosis and axonal degeneration 1–3 . It typically manifests in young adults and follows a variable clinical course, often with periods of relapse and remission. Synaptic loss has recently emerged as a key pathological feature of MS, contributing to irreversible disease progression and long-term neurological deficits 4,5 . This degenerative synaptic pathology is thought to arise when autoimmune cells infiltrate the CNS by crossing the blood-brain barrier (BBB) and attack the myelin sheath, leading to demyelination and subsequent inflammatory responses. These processes trigger glial activation, cytokine release, and excitotoxic damage to synapses, resulting in widespread synaptic density loss 6–10 . Specifically, the inflammatory milieu within the CNS is exacerbated by activated macrophages and T cells, which secrete a variety of cytokines and other soluble mediators. These substances compromise the structural integrity of the BBB, facilitating an increased infiltration of immune cells and pro-inflammatory cytokines into the neural tissue 11 . Concurrently, activated astrocytes contribute to the pathological landscape by increasing the production of reactive oxygen species (ROS), which further perpetuate cellular stress and neuronal damage 12 . This cascade of biochemical and cellular events creates a highly toxic environment that directly impairs neuronal function and leads to the progressive loss of synaptic density, critically undermining neural connectivity and function ( Fig. 1 ). The resultant synaptic degradation is a primary contributor to the cognitive decline and physical disability observed in MS patients, underscoring the complex interplay between neuroinflammation and neurodegeneration 13,14 . Experimental autoimmune encephalomyelitis (EAE), the most widely used animal model of MS, recapitulates several key features of the human disease, including neuroinflammation, demyelination, and axonal damage 15–17 . In EAE mice, excitatory synapses have been shown to destabilize and degrade, disrupting neuronal connectivity and promoting disease progression 18 . Post-mortem human studies similarly demonstrate substantial synaptic loss in various regions of the brain through immunohistochemical analyses 8,19–22 . Notably, recent evidence suggests that synaptic alterations occur independently of demyelination and axonal degeneration, with synaptic loss detected in the grey matter of MS brains and EAE models even before clinical symptoms or disease onset 23,24 . These findings highlight the pivotal role of synaptic loss in the pathogenesis of MS and suggest that effective synaptic biomarkers could be crucial for both diagnosis and the development of novel therapeutic strategies. Currently, magnetic resonance imaging (MRI) is the gold-standard diagnostic tool for MS and disease monitoring, providing detailed structural information on demyelinating lesions and brain atrophy 25,26 . However, MRI is limited in its ability to capture early molecular and cellular changes, such as synaptic loss, which drive disease progression. Additionally, MRI-derived measures often correlate poorly with clinical symptoms, particularly in progressive stages of MS 27 . These clinical challenges highlight the need for complementary imaging methods to investigate synaptic pathology and improve our understanding of MS. Positron emission tomography (PET), on the other hand, offers unique advantages for studying biochemical and cellular processes in vivo . In the context of MS, [ 18 F]FDG remains the most widely applied PET radiopharmaceuticals in clinical settings, primarily reflecting regional glucose metabolism and neuronal dysfunction 28 . Other PET radiopharmaceuticals such as those targeting translocator protein 18 kDa (TSPO) for microglial activation (e.g., [ 11 C]PK11195, [ 18 F]DPA714) and the amyloid imaging agent [ 11 C]PiB as a surrogate measure of myelin content have shown some promise in clinical research in MS 29,30 . Despite these advances, quantifying neuronal and synaptic integrity in MS remains a critical unmet need in understanding disease progression and treatment response. Among the molecular targets for PET imaging, synaptic vesicle glycoprotein 2A (SV2A) has been validated as a robust biomarker of synaptic density due to its ubiquitous expression in presynaptic terminals and critical role in neurotransmitter release 4,31 . Recent advancements in SV2A PET tracers, such as [ 11 C]UCB-J, [ 18 F]SynVesT-1/2, and [ 18 F]SDM-16 have enabled highly specific and sensitive quantification of synaptic density in various neurological disorders, including Alzheimer’s disease 22,32–39 . Despite their promising attributes, to our knowledge SV2A PET radiotracers have not been investigated in MS animal models or patients. Here, for the first time, we applied PET imaging to quantify cerebral SV2A levels in the EAE mouse model of MS. Given the established EAE pathology in the spinal cord and the potential of SV2A PET tracers for imaging spinal damage, we also assessed changes in SV2A density within the rodent spinal cord 19,40,41 . Post-mortem SV2A measurements in mouse brains were conducted to corroborate the PET imaging findings. Finally, we initiated a preliminary exploratory study of SV2A PET imaging in human MS patients using [ 11 C]UCB-J, the first SV2A radiopharmaceutical translated for clinical research ( Fig. 1 ) 33,42 . MATERIALS AND METHODS Animals and Experimental Design All experiments were conducted in accordance with the guidelines established by the institutional animal care and use committee at the Centre for Addiction and Mental Health (CAMH). EAE was induced in C57BL/6J mice (n = 18) via subcutaneous immunization with Myelin Oligodendrocyte Glycoprotein 35-55 (MOG 35-55 ) peptide in complete Freund's adjuvant, followed by intraperitoneal pertussis toxin injections using a commercially available EAE induction kit (Hooke Laboratories, catalog number EK-2110). Mice (9–13 weeks old) were group-housed in temperature- and humidity-controlled environments with food and water. They were maintained under a 12-hour light/dark cycle to minimize stress prior to and during the experimental period. Clinical EAE scores were recorded daily from the day of immunization. Mice were observed for the onset of clinical signs, with typical disease development beginning between 9- and 14-days post-immunization. Experiments were then conducted when the mice reached an EAE score of 3 or higher in this study. Naïve controls (n = 18) were subjected to the same handling and experimental conditions as the EAE group but did no receive any immunization or injections. Euthanasia was performed at the experimental endpoint for histological analysis. The expected clinical progression followed a typical chronic-relapsing EAE course, characterized by tail limpness, hind limb weakness, and eventual partial recovery, with clinical scores ranging from 0 to 5, where 0 indicates no clinical symptoms and 5 represents severe paralysis or moribundity. Human Subjects This study included five individuals with multiple sclerosis (MS) and six aged matched healthy controls ( Table 1 ). MS participants were aged 18-60, met the revised McDonald criteria for a definite diagnosis, and had an Expanded Disability Status Scale (EDSS) score between 0 and 7.5. Healthy controls were aged 18-65, matched to MS participants for age, and had normal physical exams, EKG, and blood tests. Exclusion criteria for all participants included prior/current radiation exposure exceeding FDA limits, blood donation within eight weeks, MRI contraindications, other neurological disorders mimicking MS, pregnancy/lactation, chronic corticosteroid/immunosuppressant use, active/chronic infections, progressive multifocal leukoencephalopathy, renal impairment, and bleeding disorders/anticoagulant use. The study protocol was approved by the Yale University Human Investigation Committee and the Yale New Haven Hospital Radiation Safety Committee, and adhered to 45 CFR 46, the US federal regulations for human research protection. All participants provided written informed consent after receiving a thorough explanation of the study procedures ( Fig. 1 ). Table 1. Demographic and clinical characteristics of study participants. Summary of participant characteristics for the healthy control and MS groups. The MS cohort includes 4 participants with relapsing-remitting MS (RRMS) and 1 with secondary progressive MS (SPMS). Data are presented as mean (SD) with range in parentheses where applicable. MS Small-Animal PET Scanning with [ 18 F]SynVesT-1 [ 18 F]SynVesT-1 was synthesized following previously published methods 34,35,43 . Dynamic PET/computed tomography (CT) imaging was performed using [ 18 F]SynVesT-1 in EAE mice (n = 18) and their corresponding naïve controls (n = 18). All mouse imaging studies were conducted in accordance with the guidelines established by the institutional animal care and use committees at CAMH. Mice were anesthetized using isoflurane in O₂ (4%, 2 L/min for induction; 1.5–2%, 1 L/min for maintenance) for lateral tail-vein catheterization and subsequently transferred to a nanoScan PET/CT scanner (Mediso, Budapest, Hungary) using a quad-mice pallet. Anesthesia was maintained throughout the imaging session, with body temperature and respiratory parameters monitored closely for each mouse. A scout CT scan was performed for positioning the PET field of view (FOV), followed by a material map CT acquisition for attenuation and scatter corrections using a segmented material map and for PET/CT co-registration. A bolus injection of [ 18 F]SynVesT-1 was administered via the tail-vein catheter, and dynamic PET images were acquired over a 90-minute period to assess tracer uptake and distribution. Human PET Imaging Study with [ 11 C]UCB-J [ 11 C]UCB-J was synthesized following previously published methods 33 . All participants underwent a single [ 11 C]UCB-J dynamic PET scan using the Siemens High Resolution Research Tomograph (HRRT; Siemens, Medical Solutions, Knoxville, TN, USA) with a reconstructed image resolution of approximately 3 mm. PET imaging procedures were conducted as previously described 44,45 . All participants also underwent T1-weighted magnetic resonance imaging (MRI) on a 3-T whole-body scanner (Trio, Siemens) for subsequent co-registration with the PET images. PET images were aligned to individual MR images through rigid registration. Subsequently, individual MR images were normalized to Montreal Neurological Institute space using affine linear and non-linear registration within the BioImage Suite 2.5 software. Regions of interest (ROIs) were then extracted from the automated anatomic labeling (AAL) template. Image Processing and Analysis for Rodent Scans A static image of the entire 90-minute emission acquisition was reconstructed using the manufacturer’s proprietary iterative three-dimensional (3D) algorithm (six subsets and four iterations) for co-registration with the CT scan, while dynamic images were reconstructed using both 2D-filtered back projection (FBP) and 3D-iterative (six subsets and four iterations) algorithms. The analytically reconstructed image was used for regional brain time-activity curve (TAC) extraction and the iteratively reconstructed image was used for manual cardiac blood pool TAC by manually drawing a ROI over the left ventricle of the heart, guided by the CT image, which was then used to obtain radiometabolite-corrected image-derived input functions (IDIF) in plasma based on a recent publication by Bertoglio 2022 in C57BL/6J mice. The dynamic dataset consisted of 39 frames: 3 × 5s, 3 × 15s, 3 × 20s, 7 × 60s, 17 × 180s, and 6 × 300s. Brain regions of interest (ROIs) for PET imaging were defined by co-registering PET images with a mouse brain MRI atlas 46 . The standardized uptake values (SUVs) were calculated by normalizing the regional radioactivity to the injected dose and body weight of each mouse. The standardized uptake value ratio (SUVR) was further calculated by dividing the SUV of the ROI by the SUV of a reference region. One-tissue compartmental model (1TCM) and Logan plot with plasma IDIF were used for kinetic modeling in PMOD (ver. 4.203, Zurich, Switzerland). The distribution volume ( V T ) was reported with radiometabolite-correction as described previously 47 . The distribution volume ratio (DVR) was derived using the simplified reference tissue model 2 (SRTM2). DVR IDIF was then further evaluated by dividing the IDIF-derived V T of a ROI with IDIF-derived V T of a reference region using 1TCM. Voxel-wise comparisons of spatially between naïve and EAE mouse groups were conducted using MATLAB R2024b (MathWorks) and Statistical Parametric Mapping (SPM12). Logan Plot derived V T maps were resliced and two-sample Student’s t-tests were performed using 1 –1 contrast with no explicit masking. Statistical significance at each voxel was defined as p < 0.001 and corrected for multiple comparisons using family-wise error (FWE) correction. The resulting t-maps were overlaid onto the MRI brain mouse template for visualization. Quantitative Analysis for Human Scan Voxel-wise kinetic analysis was performed using the one-tissue compartment model (1TCM) 44 and the metabolite-corrected arterial plasma curve to generate parametric images of V T . V T represents the tissue-to-plasma concentration ratio at equilibrium and reflects both specific and non-specific binding. 18 anatomically defined ROIs were applied to the parametric images using the combined transformations derived from the template-to-PET image space. Volume-weighted averages were also calculated for entire cortical ROIs, encompassing the frontal, temporal, parietal, and occipital lobes, as well as the insula and cingulate cortex. In Vitro Autoradiography using [ 18 F]SynVesT-1 Brain tissue was freshly extracted from anesthetized mice (under isoflurane) and immediately frozen at –80°C until cryostat sectioning. Frozen brain tissues were embedded in Tissue-Tek O.C.T. (Sakura Finetek, Tokyo, Japan). Serial 10 µm-thick sagittal sections of the left hemisphere were generated using a Cryostar NX50 cryostat (Thermo Fisher Scientific, Runcorn, UK) and stored at –80°C until receptor binding assays were performed. Autoradiography (ARG) was performed on brain sections from 6 naïve/EAE mice. Brain slices were incubated with [ 18 F]SynVesT-1 (10 µCi/mL). Blocking was performed using 200 µM levetiracetam (LEV), a known antiepileptic drug targeting SV2A 48 . Following incubation, sections were rinsed twice for 1 minute in rinse buffer at 4°C, followed by a 10-second rinse in ice-cold distilled water. Slides were dried under airflow for 5 minutes before being exposed to a storage phosphor (SR) screen for 30 minutes. Imaging was performed using an Amersham Typhoon™ Biomolecular Imager (GE Healthcare, MA, USA) at 25µm resolution. For quantification, 10 [ 18 F]SynVesT-1 standards were counted using a gamma counter (PerkinElmer, Waltham, MA, USA) and decay-corrected to the exposure time. Specific binding was calculated by subtracting nonspecific binding from total binding, with results expressed in µCi/mg based on the [ 18 F]SynVesT-1 calibration standards. Regional quantification was performed using MCID Core 7 (Interfocus Imaging Ltd.). Statistical Analysis Data were analyzed using GraphPad Prism (version 10). Multiple t-tests were used to compare whole-brain and regional mean V T values, SUVR and DVR between naïve and EAE mice for [ 18 F]SynVesT-1. Two-tailed, unpaired Student’s t-tests were used to compare between MS and healthy control (HC) participants for [ 11 C]UCB-J, respectively. Due to the small sample size, no correction for multiple comparisons was applied. ARG data were analyzed using Student’s t-tests to compare tracer uptake within the same animals under different conditions. RESULT PET Imaging Reveals Decreased [ 18 F]SynVesT-1 Brain Uptake in EAE Mice All naïve and EAE mice received a single injection of [ 18 F]SynVesT-1 (7.03 ± 0.882 MBq). The PET images showed visibly lower tracer uptake in EAE mice compared to naïve controls ( Figure 2A ). This reduction was also evident in the TACs, where all brain regions displayed decreased SUV values, particularly notable during the first 30 minutes post-injection. To further validate these observations, we conducted SUVR analysis using the amygdala as the reference region due to its relatively stable uptake in both EAE and naïve mice ( Supplementary Fig. 1 ). A significant decrease in SUVR was observed across all major selected brain regions during all imaging time windows (40-70 mins, 60-90 mins, 50-80 mins, Supplementary Fig. 1B , D , F ). These results significantly correlated with the DVR values estimated by SRTM2 using the amygdala as the reference region ( Supplementary Fig. 1C , E , G ). We also evaluated the V T values to further assess [ 18 F]SynVesT-1 binding in both EAE and naïve mice. Satisfyingly, the TACs were well-quantified by the 1TCM generated through population-based radiometabolite correction as previously described ( Figure 2B ). 47 Decrease in Synaptic Density in EAE Mice Through PET Imaging To quantify V T value, we conducted Logan Plot and 1TCM. As the radiometabolite correction and whole-blood-to-plasma ratio correction were based on a population-based method, assuming no influence of EAE induction on radiotracer metabolism, we also calculated the V T value without using a radiometabolite correction method. Regardless of the correction method used, there was a strong and significant correlation of V T generated by Logan Plot and 1TCM ( Fig. 3A , Supplementary Fig. 2A ). V T generated by 1TCM and Logan Plot using radiometabolite correction correlated well with each other and did not interfere with the comparison of V T generated from either kinetic modelling ( Fig. 3C, D ). We also compared the V T using the Logan Plot with two different t* and observed no significant deviation ( Supplementary Fig 2B ). Notably, the V T generated by both kinetic modelling using radiometabolite-correction exhibited the highest correlation R 2 value. Based on this observation, we generated an average V T image using Logan Plot with t* set at 9 mins and with radiometabolite correction ( Fig. 3B ). There was an observable global decrease in tracer uptake in EAE mice, which was confirmed by a voxel-wise t -statistics analysis ( Supplementary Fig. 3 ). We then compared regional V T values across various brain regions, selected based on their relevance in EAE and MS 49,50 . Using both kinetic models, we observed a statistically significant reduction in [ 18 F]SynVesT-1 uptake across all analysed brain regions ( Fig. 3C, D ). Overall, V T generated by Logan Plot was significantly reduced in the caudate putamen (-25.2%, p < 0.0001), frontal cortex (-25.9%, p < 0.0001), nucleus accumbens (-24.7%, p < 0.0001), hippocampus (-26.5%, p < 0.0001), thalamus (-26.7%, p < 0.0001), cerebral cortex (-26.6%, p < 0.0001), and whole brain (-27.9%, p < 0.0001) ( Supplementary Table 1 ). Notably, we also compared their K 1 values estimated by 1TCM and observed a significant decrease in all analysed regions (Supplementary Fig. 4). This may suggest that the decrease in V T could be due to altered cerebral blood flow in EAE mice. However, using the amygdala as the reference region, there was significantly decreased tracer uptake in terms of SUVR in the regions examined, except in the accumbens and cerebral cortex, where a non-significant trend for decrease was observed. The strong correlation between SUVR and DVR derived from SRTM2 further supports the decrease in tracer uptake and binding in EAE mice ( Supplementary Fig. 1 ). [ 18 F]SynVesT-1 PET Imaging Reveals Synaptic Density Loss in the Spinal Cord in EAE Mice We then explored the potential of using PET imaging to assess SV2A level changes in the spinal cord in EAE mice. [ 18 F]SynVesT-1 uptake in the spinal cord of EAE mice was predominantly reduced in the cervical and lumbar regions, both visually and quantitatively ( Fig. 4A, B ). Similar to the cerebral regions, the 1TCM model fits well in all spinal cord regions. The radiometabolite-corrected V T value was then estimated using 1TCM and compared between naïve and EAE conditions. A significant reduction in V T uptake was observed in the cervical, thoracic, and lumbar regions, with the sacral region remaining relatively stable and unaffected ( Fig. 4C ). Consequently, we used the sacral region as the reference region and calculated an IDIF-derived DVR. Our findings revealed a significant decrease in DVR in both the cervical (-11.8%, p < 0.05) and lumbar (-18.4%, p < 0.05) regions, with a decreasing trend observed in the thoracic region. Notably, we compared DVR values derived from SRTM2 and IDIF, finding a significant correlation and similar trends in significance ( Supplementary Fig. 5 ). Postmortem Autoradiography Analysis Corroborates SV2A Reduction in EAE Mice [ 18 F]SynVesT-1 revealed significant reductions in specific binding in the postmortem sagittal brain sections of EAE mice using ARG (-8.53%, p < 0.05) ( Fig. 5A, B ). The binding was also specific to the SV2A target, achieving 84.4 ± 0.431% specificity ( Supplementary Fig. 6 ). Consistent with in vivo PET imaging results, a significant reduction in [ 18 F]SynVesT-1-specific binding was observed in the cerebellum (-20.7%, p < 0.001), frontal cortex (-15.9%, p < 0.05), and midbrain (-9.99%, p < 0.05) ( Fig. 5C ). While a decrease in tracer uptake was also noted in the hippocampus, the reduction did not reach statistical significance, likely due to the smaller sample size utilized ( Fig. 5C ). PET Imaging Reveals Decreased Brain Uptake in MS Patients Building on promising results from preclinical animal studies, we also compared our findings with preliminary human data in MS patients using [ 11 C]UCB-J, the first SV2A tracer employed in clinical research. The study involved five individuals diagnosed with MS and six healthy controls (HC). All participants received a single injection of [ 11 C]UCB-J (509 ± 188 MBq). There was no significant difference in injected doses between groups (MS: 571 ± 190 MBq; HC: 458 ± 187 MBq; p = 0.35 ) ( Table 1 ). Arterial input functions were obtained in all participants (5 MS, 6 HC) for V T measurement ( Fig. 6 ). The primary study revealed a statistically significant 16.6% reduction in [ 11 C]UCB-J binding ( V T ) in the cortical regions of MS participants compared to HC ( p = 0.043 ), suggesting widespread loss of SV2A and synaptic density ( Fig. 6B ). Exploratory analyses demonstrated an overall decrease in [ 11 C]UCB-J binding across most examined regions in MS participants, with statistically significant reductions observed in the caudate (-25.6%, p = 0.026 ), insula (-16.1%, p = 0.044 ), ventral striatum (-16.8%, p = 0.028 ), hippocampus (-18.5%, p = 0.04 ), frontal cortex (-16.3%, p = 0.048 ), parietal cortex (-17.1%, p = 0.026), and occipital cortex (-18.6%, p = 0.020 ) ( Fig. 6C ). In addition, we examined regional brain volumes, but no significant differences were observed between groups ( Supplementary Fig. 7 ). It is important to note that these preliminary results were not corrected for multiple comparisons due to the small sample size. Discussion This study assessed the potential of using the SV2A PET radiopharmaceutical [ 18 F]SynVesT-1 to image synaptic density loss in EAE mice at clinically relevant stages of disease progression. To our knowledge, this work provides the first evidence of tracking SV2A levels in EAE mice using a non-invasive imaging tool. Notably, the findings from the preclinical animal model have also successfully translated to human MS patients where synaptic density changes were also detectable. Synaptic density loss is a key hallmark of MS 18,49,50 . Despite recent focus on studying synaptic density loss in EAE mice, no effective in vivo imaging radiotracer for detecting these changes has been reported until now. In this study, we demonstrated a significant global reduction in SV2A levels in EAE mice using [ 18 F]SynVesT-1 PET imgiang, with nearly 30% loss in all analyzed regions, reinforcing the role of synaptic pathology in MS which can be captured in vivo . Satisfyingly, our data is largely consistent with previously reported V T values in healthy mice, showing similar V T across brain regions with only small differences observed 51 . Importantly, the inflammatory environment in EAE mice may lead to modifications in perfusion dynamics and changes in cerebral blood flow, potentially resulting in altered tracer uptake 52,53 . This is also confirmed in our imaging study, which demonstrated a decrease in the K 1 value of [ 18 F]SynVesT-1 uptake in EAE mice. Given the challenges in accurately quantifying SV2A levels based solely on V T analysis in EAE mice, we further evaluated tracer uptake in the target of interest using a reference region. Interestingly, we found that the amygdala displays the most stable tracer uptake profile between naïve and EAE mice, making it a suitable reference region for SUVR calculation despite its altered kinetic characteristics ( Supplementary Fig. 4 ). Thus, since both SUVR and SRTM-2-derived DVR correlates well ( Supplementary Fig. 5 ), we simplified the kinetic quantification analysis of [ 18 F]SynVesT-1 uptake in EAE mice by calculating SUVR, and observed a global decrease in tracer uptake in the EAE brain. Nonetheless, postmortem autoradiography assays confirmed a significant decrease in tracer uptake in general, supporting the reliability of PET-based SV2A quantification in EAE mice. Nonetheless, [ 18 F]SynVesT-1 was specific to SV2A and blocked by LEV in EAE mice brain 54 . Although an insignificant decrease was observed in the hippocampus region in the ARG study, this was most likely attributed to the smaller sample size used in the experiment. The varying kinetics observed in PET imaging may also play a role in this discrepancy, as previously reported with other radiotracers 55 . As SV2A PET tracers have recently been employed as non-invasive tools to image spinal cord damage in various conditions in mice 41,56 , we also evaluated [ 18 F]SynVesT-1 PET imaging in the EAE spinal cord and demonstrated a significant reduction in SV2A levels in both cervical and lumbar sections. As expected, the uptake of tracers in the spinal cord regions was generally lower than in the cerebral regions, where we found a 2-3 fold difference between naïve and EAE mice, in agreement with previous reports 56,57 . Nonetheless, the decrease in SV2A levels in the EAE spinal cord was not as pronounced as in the brain, where we observed a 10-20% loss in the cervical and lumbar regions. Importantly, previous research has pinpointed EAE pathology in the lower sections of the spinal cord, including the lumbar region 58 . This area has also been successfully imaged using [ 18 F]SynVesT-1 PET, revealing significant changes in SV2A in the lumbar section. Furthermore, we conducted SV2A PET imaging in human MS patients using the first SV2A PET radiopharmaceutical, [ 11 C]UCB-J 33,42 . The primary findings indicated a widespread loss of SV2A and synaptic density, with a statistically significant 16.6% reduction in [ 11 C]UCB-J binding in the cortical regions of MS participants compared to healthy controls. Notably, this decreasing trend in SV2A binding was observed across all analyzed brain regions, underscoring the pervasive nature of synaptic degradation in MS and the ability of SV2A PET imaging in capturing synaptic denisty loss in MS patients. Some regions, such as the caudate and frontal cortex, reached statistical significance, highlighting areas of particularly pronounced synaptic loss. Although our study did not specifically match participants by sex, previous research suggests no significant sex differences in [ 11 C]UCB-J binding 59,60 . This finding supports the generalizability of our results across both sexes, particularly regarding the small cohort of this human study. There has been growing therapeutic effort aimed at restoring synaptic density loss in MS 8,61,62 . Considering the widespread reduction of synaptic density in MS, the use of a non-invasive biomarker like SV2A PET imaging, which is distributed across brain and gray matter, will offer crucial insights into studying MS and directing therapeutic strategies. Future research should investigate longitudinal changes of SV2A in EAE mice. Due to possible changes in tracer’s pharmacokinetics, conducting a model-based radiometabolite study is also advised. Conclusion Using PET imaging in EAE mice and human MS patients, we successfully detected significant reductions of SV2A in MS and related animal models. Our findings demonstrate that SV2A PET can reliably quantify presynaptic alterations in vivo in a preclinical MS model and a clinical research study. The observed tracer uptake patterns emphasize the potential of SV2A PET for detecting diffuse synaptic pathology. Collectively, these results support the use of SV2A PET as a non-invasive imaging tool for monitoring disease progression and evaluating therapeutic interventions in MS. Declarations Animal Ethics Statement: All animal procedures were approved by the CAMH animal Care Committee (ACC) and conducted in accordance with institutional guidelines and the relevant national regulations for the care and use of laboratory animals. References Stys, P. K. Axonal degeneration in multiple sclerosis: is it time for neuroprotective strategies? Ann. Neurol. 55 , 601–603 (2004). Stys, P. K. et al. New views on the complex interplay between degeneration and autoimmunity in multiple sclerosis. Front. Cell. Neurosci. 18 , (2024). 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[18F]FDG mouse brain PET imaging: absolute quantification of regional cerebral glucose utilization as compared with 2-[14C]DG autoradiography. Int. Congr. Ser. 1265 , 255–261 (2004). Bertoglio, D. et al. Synaptic Vesicle Glycoprotein 2A Is Affected in the Central Nervous System of Mice with Huntington Disease and in the Brain of a Human with Huntington Disease Postmortem. J. Nucl. Med. Off. Publ. Soc. Nucl. Med. 63 , 942–947 (2022). Lambeng, N., Gillard, M., Vertongen, P., Fuks, B. & Chatelain, P. Characterization of [3H]ucb 30889 binding to synaptic vesicle protein 2A in the rat spinal cord. Eur. J. Pharmacol. 520 , 70–76 (2005). Xin, L. et al. Impairment of spinal CSF flow precedes immune cell infiltration in an active EAE model. J. Neuroinflammation 21 , 272 (2024). Michiels, L. et al. Synaptic density in healthy human aging is not influenced by age or sex: a 11C-UCB-J PET study. NeuroImage 232 , 117877 (2021). Toyonaga, T. et al. The regional pattern of age-related synaptic loss in the human brain differs from gray matter volume loss: in vivo PET measurement with [11C]UCB-J. Eur. J. Nucl. Med. Mol. Imaging 51 , 1012–1022 (2024). Zhai, D. et al. Small-molecule targeting AMPA-mediated excitotoxicity has therapeutic effects in mouse models for multiple sclerosis. Sci. Adv. 9 , eadj6187. Poon, M. M. et al. Targeting the muscarinic M1 receptor with a selective, brain-penetrant antagonist to promote remyelination in multiple sclerosis. Proc. Natl. Acad. Sci. 121 , e2407974121 (2024). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryInformationSV2AMS5.27.docx Supplementary Information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6769442","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":464073306,"identity":"03cf37d2-1108-419f-a738-b8d27697b808","order_by":0,"name":"Chao Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYDACCTB5AIiZD0AZxGthSyBZC48BcVr4Zzc/fMDw546cOf+ajx9+5txh4G8noE3izjFjAwaeZ8aWM95uluzd9oxB4kwCfi0GEglmEgwShxM33Di7jYF322EGAwaCWtK/STAYHK7fcOPMM8a/IC38DwhpyQHaknA4weB8Dxsz2BYJArZI3MgpNkg4cNhwww02Y2nZbYd5JG4QsIV/RvrGBx/+HJY3OH/44ce32w7L8fcTsAUMwGqg7uEhQj3cvgMkKB4Fo2AUjIIRBQAVTUnl8/nHiAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2179-8096","institution":"Centre for Addiction and Mental Health \u0026 University of Toronto","correspondingAuthor":true,"prefix":"","firstName":"Chao","middleName":"","lastName":"Zheng","suffix":""},{"id":464073307,"identity":"dc5777ce-81cb-47b5-89f4-1cda8f9a7a36","order_by":1,"name":"Pou Hong Justin Chia","email":"","orcid":"","institution":"Centre for Addiction and Mental Health \u0026 University of Toronto","correspondingAuthor":false,"prefix":"","firstName":"Pou","middleName":"Hong Justin","lastName":"Chia","suffix":""},{"id":464073308,"identity":"b1f75583-b1e1-4aa0-951a-0a023df5a790","order_by":2,"name":"Takuya Toyonaga","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Takuya","middleName":"","lastName":"Toyonaga","suffix":""},{"id":464073309,"identity":"824e0394-a5e2-4d1b-8ea6-2902616a9b95","order_by":3,"name":"Junchao Tong","email":"","orcid":"","institution":"Centre for Addiction and Mental Health","correspondingAuthor":false,"prefix":"","firstName":"Junchao","middleName":"","lastName":"Tong","suffix":""},{"id":464073310,"identity":"34780277-6c7e-4d30-b9a7-db8bde69585b","order_by":4,"name":"Hannah Le","email":"","orcid":"","institution":"Centre for Addiction and Mental Health","correspondingAuthor":false,"prefix":"","firstName":"Hannah","middleName":"","lastName":"Le","suffix":""},{"id":464073311,"identity":"d2874ecf-f3a3-4d24-bf06-d87ed4c1d3e7","order_by":5,"name":"Mark Dias","email":"","orcid":"","institution":"Yale University","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Dias","suffix":""},{"id":464073312,"identity":"21450315-0d30-40ea-b955-1a7cf8c82618","order_by":6,"name":"Amanda J. 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(A) \u003c/strong\u003eIllustration of the cellular mechanisms contributing to synaptic damage in MS. The diagram shows cytokines and inflammatory cells infiltrating the CNS and triggering demyelination. This leads to the activation of astrocytes and the production of ROS, which contribute to oxidative stress, more demyelination, and neuronal dysfunction. The loss of myelin impairs axonal transport and synaptic vesicle recycling, possibly reducing SV2A expression and neurotransmitter release, ultimately leading to synaptic degeneration.\u003cstrong\u003e (B)\u003c/strong\u003e Experimental workflow for SV2A imaging in EAE mice. EAE was induced in C57BL/6J mice and scanned only upon reaching a clinical score of ≥ 3 (n = 18/group). Following imaging, brains were extracted, sectioned, and subjected to autoradiography (ARG) for SV2A quantification (n = 6/group). Representative PET and ARG images of SV2A binding in naïve and EAE mouse are shown. \u003cstrong\u003e(C)\u003c/strong\u003eWorkflow for the human imaging study. Healthy controls and MS patients underwent screening and eligibility assessment before PET imaging. Imaging included quality control and patient-based radiometabolite correction before quantification of SV2A binding.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/116d64c791efa370638d468f.png"},{"id":83837649,"identity":"85c6dfaa-64d2-44d2-90bd-f03ef084e591","added_by":"auto","created_at":"2025-06-03 13:25:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":147975,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[¹⁸F]SynVesT-1 uptake is reduced in EAE mice. (A) \u003c/strong\u003eRepresentative CT and PET SUV (0-90 mins) images of [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 in naïve and EAE mouse and the overlaid image with an MRI mouse brain atlas. Scale bar represents SUV value. \u003cstrong\u003e(B)\u003c/strong\u003e TACs for multiple selected brain regions, including the frontal cortex, midbrain, hippocampus, thalamus, cerebellar cortex, and whole brain. Solid lines represent 1TCM-model fitting curve.\u003cstrong\u003e \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/6ec7cd7d37db60a996116a74.png"},{"id":83838225,"identity":"7927286d-dcb0-44fb-b8e0-1847507a87b0","added_by":"auto","created_at":"2025-06-03 13:33:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":175439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[¹⁸F]SynVesT-1 binding is reduced in EAE mice. (A) \u003c/strong\u003eCorrelation between \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values estimated by the radiometabolite-corrected Logan plot and the 1TCM across brain regions, showing a strong linear relationship (n=4, R² = 1, p \u0026lt; 0.0001). Blue line represents the line of identity. \u003cstrong\u003e(B)\u003c/strong\u003e Averaged parametric images of \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e\u003cem\u003e \u003c/em\u003ederived from radiometabolite-corrected Logan Plot, shown in coronal, sagittal, and axial views. The color scale represents \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values (mL/cm³). \u003cstrong\u003e(C)\u003c/strong\u003e Regional \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values derived from the Logan plot, demonstrating a significant reduction in [¹⁸F]SynVesT-1 binding in multiple brain regions of EAE mice compared to naïve controls (n = 18/group). \u003cstrong\u003e(D)\u003c/strong\u003e Regional \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values estimated by the 1TCM, confirming reduced tracer binding in EAE mice (n = 18/group). Data are presented as mean ± SD; \u003cem\u003e****p \u0026lt; 0.0001\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/6ef7486b5af72e92c6e6c9b2.png"},{"id":83837654,"identity":"5794882d-6323-4b18-9366-531d61347d61","added_by":"auto","created_at":"2025-06-03 13:25:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":220335,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[¹⁸F]SynVesT-1 uptake is reduced in the spinal cord of EAE mice. (A) \u003c/strong\u003eRepresentative axial view of PET images of [¹⁸F]SynVesT-1 uptake in the cervical, thoracic, lumbar and sacral spinal cord regions of naïve and EAE mice. The color scale represents SUV values. \u003cstrong\u003e(B)\u003c/strong\u003e TACs of [¹⁸F]SynVesT-1 SUV in the cervical, thoracic, and lumbar spinal cord regions over 90 min, showing observable reduced tracer uptake in EAE mice. Solid line represents 1TCM fitting curve. \u003cstrong\u003e(C)\u003c/strong\u003e Regional \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values derived from radiometabolite-corrected 1TCM, demonstrating significant reductions of tracer uptake in the cervical, thoracic and lumbar regions of EAE mice compared to naïve controls. \u003cstrong\u003e(D)\u003c/strong\u003e Regional DVR values in the spinal cord. DVR was calculated by dividing \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e of the region of interest by \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e of the sacral region. Data are presented as mean ± SD (n = 18/group; one EAE mouse was excluded from SUVR and DVR calculations due to being a significant outlier); \u003cem\u003e*p \u0026lt; 0.05, ***p \u0026lt; 0.001\u003c/em\u003e, ns = not significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/b720cb15a39b288cead0af72.png"},{"id":83837650,"identity":"cef3cb64-abe0-4e6c-8b10-61c35edcfa44","added_by":"auto","created_at":"2025-06-03 13:25:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98176,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePost-mortem quantification reveals reduced SV2A binding in EAE mice. (A) \u003c/strong\u003eRepresentative autoradiography images of [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 binding in the sagittal brain sections of naïve and EAE mice. Scale bar represents binding in µCi/g.\u003cstrong\u003e (B)\u003c/strong\u003e Regional analysis of specific binding demonstrates a significant reduction in the cerebellum, frontal cortex, midbrain and whole brain of EAE mice, while hippocampal binding remains unchanged (n = 6/group). \u003cem\u003e*p \u0026lt; 0.05, ***p \u0026lt; 0.001, \u003c/em\u003ens = not significant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/7461ae7ac0250350931acf39.png"},{"id":83837655,"identity":"d6f903b5-71cc-455f-ba5c-58e04d7c77cd","added_by":"auto","created_at":"2025-06-03 13:25:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":161011,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e[\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e11\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC]UCB-J PET reveals decreased SV2A tracer uptake in MS. (A) \u003c/strong\u003eComparison of [\u003csup\u003e11\u003c/sup\u003eC]UCB-J binding in the cortical regions between people with MS and HCs. \u003cstrong\u003e(B) \u003c/strong\u003eIllustration of the difference in total brain [\u003csup\u003e11\u003c/sup\u003eC]-UCB-J uptake between an MS participant and a HC\u003cstrong\u003e.\u0026nbsp; \u003c/strong\u003eThe top row shows the MRI (FLAIR and T1-weighted) and [\u003csup\u003e11\u003c/sup\u003eC]UCB-J \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e parametric PET image of a female MS participant. The bottom row displays the corresponding MRI and [\u003csup\u003e11\u003c/sup\u003eC]UCB-J \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e parametric PET image of a similarly aged HC. \u003cstrong\u003e(C) \u003c/strong\u003eComparison of [\u003csup\u003e11\u003c/sup\u003eC]UCB-J bindings in different brain regions between people with MS and HCs. Data are presented as mean ± SD. \u003cem\u003e*p ≤ 0.05\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/7658539f8f12c020948ec2a8.png"},{"id":84915559,"identity":"6983f815-56df-4272-9883-06786d9e2ffc","added_by":"auto","created_at":"2025-06-18 18:09:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2325417,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/b134cb75-8854-4e72-918d-a9932edde317.pdf"},{"id":83837652,"identity":"fc4ce4ac-b317-4499-9eae-969fa83d6912","added_by":"auto","created_at":"2025-06-03 13:25:38","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":821865,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformationSV2AMS5.27.docx","url":"https://assets-eu.researchsquare.com/files/rs-6769442/v1/2ee5e2cf73ef6e1134c2dd81.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"PET Imaging of Synaptic Density Loss in Patients with Multiple Sclerosis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMultiple sclerosis (MS) is a chronic inflammatory neurodegenerative disease of the central nervous system (CNS), characterized by demyelination, gliosis and axonal degeneration\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. It typically manifests in young adults and follows a variable clinical course, often with periods of relapse and remission. Synaptic loss has recently emerged as a key pathological feature of MS, contributing to irreversible disease progression and long-term neurological deficits\u003csup\u003e4,5\u003c/sup\u003e. This degenerative synaptic pathology is thought to arise when autoimmune cells infiltrate the CNS by crossing the blood-brain barrier (BBB) and attack the myelin sheath, leading to demyelination and subsequent inflammatory responses. These processes trigger glial activation, cytokine release, and excitotoxic damage to synapses, resulting in widespread synaptic density loss\u003csup\u003e6\u0026ndash;10\u003c/sup\u003e.\u0026nbsp;Specifically, the inflammatory milieu within the CNS is exacerbated by activated macrophages and T cells, which secrete a variety of cytokines and other soluble mediators. These substances compromise the structural integrity of the BBB, facilitating an increased infiltration of immune cells and pro-inflammatory cytokines into the neural tissue\u003csup\u003e11\u003c/sup\u003e. Concurrently, activated astrocytes contribute to the pathological landscape by increasing the production of reactive oxygen species (ROS), which further perpetuate cellular stress and neuronal damage\u003csup\u003e12\u003c/sup\u003e. This cascade of biochemical and cellular events creates a highly toxic environment that directly impairs neuronal function and leads to the progressive loss of synaptic density, critically undermining neural connectivity and function (\u003cstrong\u003eFig. 1\u003c/strong\u003e). The resultant synaptic degradation is a primary contributor to the cognitive decline and physical disability observed in MS patients, underscoring the complex interplay between neuroinflammation and neurodegeneration\u003csup\u003e13,14\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eExperimental autoimmune encephalomyelitis (EAE), the most widely used animal model of MS, recapitulates several key features of the human disease, including neuroinflammation, demyelination, and axonal damage\u003csup\u003e15\u0026ndash;17\u003c/sup\u003e. In EAE mice, excitatory synapses have been shown to destabilize and degrade, disrupting neuronal connectivity and promoting disease progression\u003csup\u003e18\u003c/sup\u003e. Post-mortem human studies similarly demonstrate substantial synaptic loss in various regions of the brain through immunohistochemical analyses\u003csup\u003e8,19\u0026ndash;22\u003c/sup\u003e. Notably, recent evidence suggests that synaptic alterations occur independently of demyelination and axonal degeneration, with synaptic loss detected in the grey matter of MS brains and EAE models even before clinical symptoms or disease onset\u003csup\u003e23,24\u003c/sup\u003e. These findings highlight the pivotal role of synaptic loss in the pathogenesis of MS and suggest that effective synaptic biomarkers could be crucial for both diagnosis and the development of novel therapeutic strategies.\u003c/p\u003e\n\u003cp\u003eCurrently, magnetic resonance imaging (MRI) is the gold-standard diagnostic tool for MS and disease monitoring, providing detailed structural information on demyelinating lesions and brain atrophy\u003csup\u003e25,26\u003c/sup\u003e. However, MRI is limited in its ability to capture early molecular and cellular changes, such as synaptic loss, which drive disease progression. Additionally, MRI-derived measures often correlate poorly with clinical symptoms, particularly in progressive stages of MS\u003csup\u003e27\u003c/sup\u003e. These clinical challenges highlight the need for complementary imaging methods to investigate synaptic pathology and improve our understanding of MS. Positron emission tomography (PET), on the other hand, offers unique advantages for studying biochemical and cellular processes \u003cem\u003ein vivo\u003c/em\u003e. In the context of MS, [\u003csup\u003e18\u003c/sup\u003eF]FDG remains the most widely applied PET radiopharmaceuticals in clinical settings, primarily reflecting regional glucose metabolism and neuronal dysfunction\u003csup\u003e28\u003c/sup\u003e. Other PET radiopharmaceuticals such as those targeting translocator protein 18 kDa (TSPO) for microglial activation (e.g., [\u003csup\u003e11\u003c/sup\u003eC]PK11195, [\u003csup\u003e18\u003c/sup\u003eF]DPA714) and the amyloid imaging agent [\u003csup\u003e11\u003c/sup\u003eC]PiB as a surrogate measure of myelin content have shown some promise in clinical research in MS\u003csup\u003e29,30\u003c/sup\u003e. Despite these advances, quantifying neuronal and synaptic integrity in MS remains a critical unmet need in understanding disease progression and treatment response. Among the molecular targets for PET imaging, synaptic vesicle glycoprotein 2A (SV2A) has been validated as a robust biomarker of synaptic density due to its ubiquitous expression in presynaptic terminals and critical role in neurotransmitter release\u003csup\u003e4,31\u003c/sup\u003e. Recent advancements in SV2A PET tracers, such as [\u003csup\u003e11\u003c/sup\u003eC]UCB-J, [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1/2, and [\u003csup\u003e18\u003c/sup\u003eF]SDM-16 have enabled highly specific and sensitive quantification of synaptic density in various neurological disorders, including Alzheimer\u0026rsquo;s disease\u003csup\u003e22,32\u0026ndash;39\u003c/sup\u003e. Despite their promising attributes, to our knowledge SV2A PET radiotracers have not been investigated in MS animal models or patients.\u003c/p\u003e\n\u003cp\u003eHere, for the first time, we applied PET imaging to quantify cerebral SV2A levels in the EAE mouse model of MS. Given the established EAE pathology in the spinal cord and the potential of SV2A PET tracers for imaging spinal damage, we also assessed changes in SV2A density within the rodent spinal cord\u003csup\u003e19,40,41\u003c/sup\u003e. Post-mortem SV2A measurements in mouse brains were conducted to corroborate the PET imaging findings. Finally, we initiated a preliminary exploratory study of SV2A PET imaging in human MS patients using [\u003csup\u003e11\u003c/sup\u003eC]UCB-J, the first SV2A radiopharmaceutical translated for clinical research (\u003cstrong\u003eFig. 1\u003c/strong\u003e)\u003csup\u003e33,42\u003c/sup\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eAnimals and Experimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were conducted in accordance with the guidelines established by the institutional animal care and use committee at the Centre for Addiction and Mental Health (CAMH). EAE was induced in C57BL/6J mice (n = 18) via subcutaneous immunization with Myelin Oligodendrocyte Glycoprotein\u003csub\u003e35-55\u003c/sub\u003e (MOG\u003csub\u003e35-55\u003c/sub\u003e) peptide in complete Freund\u0026apos;s adjuvant, followed by intraperitoneal pertussis toxin injections using a commercially available EAE induction kit (Hooke Laboratories, catalog number EK-2110). Mice (9\u0026ndash;13 weeks old) were group-housed in temperature- and humidity-controlled environments with food and water. They were maintained under a 12-hour light/dark cycle to minimize stress prior to and during the experimental period. Clinical EAE scores were recorded daily from the day of immunization. Mice were observed for the onset of clinical signs, with typical disease development beginning between 9- and 14-days post-immunization. Experiments were then conducted when the mice reached an EAE score of 3 or higher in this study. Na\u0026iuml;ve controls (n = 18) were subjected to the same handling and experimental conditions as the EAE group but did no receive any immunization or injections. Euthanasia was performed at the experimental endpoint for histological analysis. The expected clinical progression followed a typical chronic-relapsing EAE course, characterized by tail limpness, hind limb weakness, and eventual partial recovery, with clinical scores ranging from 0 to 5, where 0 indicates no clinical symptoms and 5 represents severe paralysis or moribundity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Subjects\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study included five individuals with multiple sclerosis (MS) and six aged matched healthy controls (\u003cstrong\u003eTable 1\u003c/strong\u003e). MS participants were aged 18-60, met the revised McDonald criteria for a definite diagnosis, and had an Expanded Disability Status Scale (EDSS) score between 0 and 7.5. Healthy controls were aged 18-65, matched to MS participants for age, and had normal physical exams, EKG, and blood tests. Exclusion criteria for all participants included prior/current radiation exposure exceeding FDA limits, blood donation within eight weeks, MRI contraindications, other neurological disorders mimicking MS, pregnancy/lactation, chronic corticosteroid/immunosuppressant use, active/chronic infections, progressive multifocal leukoencephalopathy, renal impairment, and bleeding disorders/anticoagulant use. The study protocol was approved by the Yale University Human Investigation Committee and the Yale New Haven Hospital Radiation Safety Committee, and adhered to 45 CFR 46, the US federal regulations for human research protection. All participants provided written informed consent after receiving a thorough explanation of the study procedures (\u003cstrong\u003eFig. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Demographic and clinical characteristics of study participants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cimg 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\" width=\"863\" height=\"324\"\u003e\u003c/strong\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eSummary of participant characteristics for the healthy control and MS groups. The MS cohort includes 4 participants with relapsing-remitting MS (RRMS) and 1 with secondary progressive MS (SPMS). Data are presented as mean (SD) with range in parentheses where applicable. MS\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSmall-Animal PET Scanning with [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 was synthesized following previously published methods\u003csup\u003e34,35,43\u003c/sup\u003e.\u0026nbsp;Dynamic PET/computed tomography (CT) imaging was performed using [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 in EAE mice (n = 18) and their corresponding na\u0026iuml;ve controls (n = 18). All mouse imaging studies were conducted in accordance with the guidelines established by the institutional animal care and use committees at CAMH. Mice were anesthetized using isoflurane in O₂ (4%, 2 L/min for induction; 1.5\u0026ndash;2%, 1 L/min for maintenance) for lateral tail-vein catheterization and subsequently transferred to a nanoScan PET/CT scanner (Mediso, Budapest, Hungary) using a quad-mice pallet. Anesthesia was maintained throughout the imaging session, with body temperature and respiratory parameters monitored closely for each mouse. A scout CT scan was performed for positioning the PET field of view (FOV), followed by a material map CT acquisition for attenuation and scatter corrections using a segmented material map and for PET/CT co-registration. A bolus injection of [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 was administered via the tail-vein catheter, and dynamic PET images were acquired over a 90-minute period to assess tracer uptake and distribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman\u003c/strong\u003e \u003cstrong\u003e\u003cspan id=\"_Toc314604649\"\u003ePET Imaging Study with \u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003e[\u003csup\u003e11\u003c/sup\u003eC]UCB-J\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e11\u003c/sup\u003eC]UCB-J was synthesized following previously published methods\u003csup\u003e33\u003c/sup\u003e.\u003csup\u003e\u0026nbsp;\u003c/sup\u003eAll participants\u0026nbsp;underwent\u0026nbsp;a single [\u003csup\u003e11\u003c/sup\u003eC]UCB-J dynamic PET scan using the Siemens High Resolution Research Tomograph (HRRT; Siemens, Medical Solutions, Knoxville, TN, USA) with a reconstructed image resolution of\u0026nbsp;approximately\u0026nbsp;3 mm.\u0026nbsp;PET imaging procedures were conducted as previously described\u003csup\u003e44,45\u003c/sup\u003e. \u0026nbsp;All participants also underwent T1-weighted magnetic resonance imaging (MRI) on a 3-T whole-body scanner (Trio, Siemens) for subsequent co-registration with the PET images. PET images were aligned to individual MR images through rigid registration. Subsequently, individual MR images were normalized to Montreal Neurological Institute space using affine linear and non-linear registration within the BioImage Suite 2.5 software. Regions of interest (ROIs) were then extracted from the automated anatomic labeling (AAL) template.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImage Processing and Analysis for Rodent Scans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA static image of the entire 90-minute emission acquisition was reconstructed using the manufacturer\u0026rsquo;s proprietary iterative three-dimensional (3D) algorithm (six subsets and four iterations) for co-registration with the CT scan, while dynamic images were reconstructed using both 2D-filtered back projection (FBP) and 3D-iterative (six subsets and four iterations) algorithms. The analytically reconstructed image was used for regional brain time-activity curve (TAC) extraction and the iteratively reconstructed image was used for manual cardiac blood pool TAC by manually drawing a ROI over the left ventricle of the heart, guided by the CT image, which was then used to obtain radiometabolite-corrected image-derived input functions (IDIF) in plasma based on a recent publication by Bertoglio 2022 in C57BL/6J mice. The dynamic dataset consisted of 39 frames: 3 \u0026times; 5s, 3 \u0026times; 15s, 3 \u0026times; 20s, 7 \u0026times; 60s, 17 \u0026times; 180s, and 6 \u0026times; 300s. Brain regions of interest (ROIs) for PET imaging were defined by co-registering PET images with a mouse brain MRI atlas\u003csup\u003e46\u003c/sup\u003e. The standardized uptake values (SUVs) were calculated by normalizing the regional radioactivity to the injected dose and body weight of each mouse. The standardized uptake value ratio (SUVR) was further calculated by dividing the SUV of the ROI by the SUV of a reference region. One-tissue compartmental model (1TCM) and Logan plot with plasma IDIF were used for kinetic modeling in PMOD (ver. 4.203, Zurich, Switzerland). The distribution volume (\u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e) was reported with radiometabolite-correction as described previously\u003csup\u003e47\u003c/sup\u003e. The distribution volume ratio (DVR) was derived using the simplified reference tissue model 2 (SRTM2). DVR\u003csub\u003eIDIF\u003c/sub\u003e was then further evaluated by dividing the IDIF-derived \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e of a ROI with IDIF-derived \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e of a reference region using 1TCM. Voxel-wise comparisons of spatially between na\u0026iuml;ve and EAE mouse groups were conducted using MATLAB R2024b (MathWorks) and Statistical Parametric Mapping (SPM12). Logan Plot derived \u003cem\u003eV\u003csub\u003eT\u003c/sub\u003e\u0026nbsp;\u003c/em\u003emaps were resliced and two-sample Student\u0026rsquo;s t-tests were performed using 1 \u0026ndash;1 contrast with no explicit masking. Statistical significance at each voxel was defined as \u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e and corrected for multiple comparisons using family-wise error (FWE) correction. The resulting t-maps were overlaid onto the MRI brain mouse template for visualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative Analysis for Human Scan\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVoxel-wise kinetic analysis was performed using the one-tissue compartment model (1TCM)\u003csup\u003e44\u003c/sup\u003e and the metabolite-corrected arterial plasma curve to generate parametric images of \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e. \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e represents the tissue-to-plasma concentration ratio at equilibrium and reflects both specific and non-specific binding. 18 anatomically defined ROIs were applied to the parametric images using the combined transformations derived from the template-to-PET image space. Volume-weighted averages were also calculated for entire cortical ROIs, encompassing the frontal, temporal, parietal, and occipital lobes, as well as the insula and cingulate cortex.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn Vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Autoradiography using [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBrain tissue was freshly extracted from anesthetized mice (under isoflurane) and immediately frozen at \u0026ndash;80\u0026deg;C until cryostat sectioning. Frozen brain tissues were embedded in Tissue-Tek O.C.T. (Sakura Finetek, Tokyo, Japan). Serial 10 \u0026micro;m-thick sagittal sections of the left hemisphere were generated using a Cryostar NX50 cryostat (Thermo Fisher Scientific, Runcorn, UK) and stored at \u0026ndash;80\u0026deg;C until receptor binding assays were performed. Autoradiography (ARG) was performed on brain sections from 6 na\u0026iuml;ve/EAE mice. Brain slices were incubated with [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 (10 \u0026micro;Ci/mL). Blocking was performed using 200 \u0026micro;M levetiracetam (LEV), a known antiepileptic drug targeting SV2A\u003csup\u003e48\u003c/sup\u003e. Following incubation, sections were rinsed twice for 1 minute in rinse buffer at 4\u0026deg;C, followed by a 10-second rinse in ice-cold distilled water. Slides were dried under airflow for 5 minutes before being exposed to a storage phosphor (SR) screen for 30 minutes. Imaging was performed using an Amersham Typhoon\u0026trade; Biomolecular Imager (GE Healthcare, MA, USA) at 25\u0026micro;m resolution. For quantification, 10 [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 standards were counted using a gamma counter (PerkinElmer, Waltham, MA, USA) and decay-corrected to the exposure time. Specific binding was calculated by subtracting nonspecific binding from total binding, with results expressed in \u0026micro;Ci/mg based on the [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 calibration standards. Regional quantification was performed using MCID Core 7 (Interfocus Imaging Ltd.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using GraphPad Prism (version 10). Multiple t-tests were used to compare whole-brain and regional mean \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values, SUVR and DVR between na\u0026iuml;ve and EAE mice for [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1. Two-tailed, unpaired Student\u0026rsquo;s t-tests were used to compare between MS and\u0026nbsp;healthy control (HC) participants for [\u003csup\u003e11\u003c/sup\u003eC]UCB-J, respectively. Due to the small sample size, no correction for multiple comparisons was applied. ARG data were analyzed using Student\u0026rsquo;s t-tests to compare tracer uptake within the same animals under different conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"RESULT","content":"\u003cp\u003e\u003cstrong\u003ePET Imaging Reveals Decreased [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 Brain Uptake in EAE Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll na\u0026iuml;ve and EAE mice received a single injection of [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 (7.03 \u0026plusmn; 0.882 MBq). The PET images showed visibly lower tracer uptake in EAE mice compared to na\u0026iuml;ve controls (\u003cstrong\u003eFigure 2A\u003c/strong\u003e). This reduction was also evident in the TACs, where all brain regions displayed decreased SUV values, particularly notable during the first 30 minutes post-injection. To further validate these observations, we conducted SUVR analysis using the amygdala as the reference region due to its relatively stable uptake in both EAE and na\u0026iuml;ve mice (\u003cstrong\u003eSupplementary Fig. 1\u003c/strong\u003e). A significant decrease in SUVR was observed across all major selected brain regions during all imaging time windows (40-70 mins, 60-90 mins, 50-80 mins, \u003cstrong\u003eSupplementary Fig. 1B\u003c/strong\u003e, \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e). These results significantly correlated with the DVR values estimated by SRTM2 using the amygdala as the reference region (\u003cstrong\u003eSupplementary Fig. 1C\u003c/strong\u003e, \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eG\u003c/strong\u003e). We also evaluated the \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values to further assess [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 binding in both EAE and na\u0026iuml;ve mice. Satisfyingly, the TACs were well-quantified by the 1TCM generated through population-based radiometabolite correction as previously described (\u003cstrong\u003eFigure 2B\u003c/strong\u003e).\u003csup\u003e47\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDecrease in Synaptic Density in EAE Mice Through PET Imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e value, we conducted Logan Plot and 1TCM. As the radiometabolite correction and whole-blood-to-plasma ratio correction were based on a population-based method, assuming no influence of EAE induction on radiotracer metabolism, we also calculated the \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e value without using a radiometabolite correction method. Regardless of the correction method used, there was a strong and significant correlation of \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e generated by Logan Plot and 1TCM (\u003cstrong\u003eFig. 3A\u003c/strong\u003e, \u003cstrong\u003eSupplementary Fig. 2A\u003c/strong\u003e). \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e generated by 1TCM and Logan Plot using radiometabolite correction correlated well with each other and did not interfere with the comparison of \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e generated from either kinetic modelling (\u003cstrong\u003eFig. 3C, D\u003c/strong\u003e). We also compared the \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e using the Logan Plot with two different t* and observed no significant deviation (\u003cstrong\u003eSupplementary Fig 2B\u003c/strong\u003e). Notably, the \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e generated by both kinetic modelling using radiometabolite-correction exhibited the highest correlation R\u003csup\u003e2\u003c/sup\u003e value. Based on this observation, we generated an average \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e image using Logan Plot with t* set at 9 mins and with radiometabolite correction (\u003cstrong\u003eFig. 3B\u003c/strong\u003e). There was an observable global decrease in tracer uptake in EAE mice, which was confirmed by a voxel-wise \u003cem\u003et\u003c/em\u003e-statistics analysis (\u003cstrong\u003eSupplementary Fig. 3\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eWe then compared regional \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003evalues across various brain regions, selected based on their relevance in EAE and MS\u003csup\u003e49,50\u003c/sup\u003e. Using both kinetic models, we observed a statistically significant reduction in [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 uptake across all analysed brain regions (\u003cstrong\u003eFig. 3C, D\u003c/strong\u003e). Overall, \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e generated by Logan Plot was significantly reduced in the caudate putamen (-25.2%, p \u0026lt; 0.0001), frontal cortex (-25.9%, p \u0026lt; 0.0001), nucleus accumbens (-24.7%, p \u0026lt; 0.0001), hippocampus (-26.5%, p \u0026lt; 0.0001), thalamus (-26.7%, p \u0026lt; 0.0001), cerebral cortex (-26.6%, p \u0026lt; 0.0001), and whole brain (-27.9%, p \u0026lt; 0.0001) (\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e). Notably, we also compared their \u003cem\u003eK\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e values estimated by 1TCM and observed a significant decrease in all analysed regions (Supplementary Fig. 4). This may suggest that the decrease in \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e could be due to altered cerebral blood flow in EAE mice. However, using the amygdala as the reference region, there was significantly decreased tracer uptake in terms of SUVR in the regions examined, except in the accumbens and cerebral cortex, where a non-significant trend for decrease was observed. The strong correlation between SUVR and DVR derived from SRTM2 further supports the decrease in tracer uptake and binding in EAE mice (\u003cstrong\u003eSupplementary Fig. 1\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e[\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 PET Imaging Reveals Synaptic Density Loss in the Spinal Cord in EAE Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe then explored the potential of using PET imaging to assess SV2A level changes in the spinal cord in EAE mice. [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 uptake in the spinal cord of EAE mice was predominantly reduced in the cervical and lumbar regions, both visually and quantitatively (\u003cstrong\u003eFig. 4A, B\u003c/strong\u003e). Similar to the cerebral regions, the 1TCM model fits well in all spinal cord regions. The radiometabolite-corrected \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e value was then estimated using 1TCM and compared between na\u0026iuml;ve and EAE conditions. A significant reduction in \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e uptake was observed in the cervical, thoracic, and lumbar regions, with the sacral region remaining relatively stable and unaffected (\u003cstrong\u003eFig. 4C\u003c/strong\u003e). Consequently, we used the sacral region as the reference region and calculated an IDIF-derived DVR. Our findings revealed a significant decrease in DVR in both the cervical (-11.8%, p \u0026lt; 0.05) and lumbar (-18.4%, p \u0026lt; 0.05) regions, with a decreasing trend observed in the thoracic region.\u0026nbsp;Notably, we compared DVR values derived from SRTM2 and IDIF, finding a significant correlation and similar trends in significance (\u003cstrong\u003eSupplementary Fig. 5\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePostmortem Autoradiography Analysis Corroborates SV2A Reduction in EAE Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 revealed significant reductions in specific binding in the postmortem sagittal brain sections of EAE mice using ARG (-8.53%, p \u0026lt; 0.05) (\u003cstrong\u003eFig. 5A, B\u003c/strong\u003e). The binding was also specific to the SV2A target, achieving 84.4 \u0026plusmn; 0.431% specificity (\u003cstrong\u003eSupplementary Fig. 6\u003c/strong\u003e). Consistent with \u003cem\u003ein vivo\u003c/em\u003e PET imaging results, a significant reduction in [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1-specific binding was observed in the cerebellum (-20.7%, p \u0026lt; 0.001), frontal cortex (-15.9%, p \u0026lt; 0.05), and midbrain (-9.99%, p \u0026lt; 0.05) (\u003cstrong\u003eFig. 5C\u003c/strong\u003e). While a decrease in tracer uptake was also noted in the hippocampus, the reduction did not reach statistical significance, likely due to the smaller sample size utilized (\u003cstrong\u003eFig. 5C\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePET Imaging Reveals Decreased Brain Uptake in MS Patients\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBuilding on promising results from preclinical animal studies, we also compared our findings with preliminary human data in MS patients using [\u003csup\u003e11\u003c/sup\u003eC]UCB-J, the first SV2A tracer employed in clinical research. The study involved five individuals diagnosed with MS and six healthy controls (HC). All participants received a single injection of [\u003csup\u003e11\u003c/sup\u003eC]UCB-J (509\u0026nbsp;\u0026plusmn;\u0026nbsp;188 MBq). There was no significant difference in injected doses between groups (MS: 571 \u0026plusmn; 190 MBq; HC: 458 \u0026plusmn; 187 MBq; \u003cem\u003ep = 0.35\u003c/em\u003e) (\u003cstrong\u003eTable 1\u003c/strong\u003e). Arterial input functions were obtained in all participants (5 MS, 6 HC) for \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e measurement (\u003cstrong\u003eFig. 6\u003c/strong\u003e). The primary study revealed a statistically significant 16.6% reduction in [\u003csup\u003e11\u003c/sup\u003eC]UCB-J binding (\u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e) in the cortical regions of MS participants compared to HC (\u003cem\u003ep = 0.043\u003c/em\u003e), suggesting widespread loss of SV2A and synaptic density (\u003cstrong\u003eFig. 6B\u003c/strong\u003e). Exploratory analyses demonstrated an overall decrease in [\u003csup\u003e11\u003c/sup\u003eC]UCB-J binding across most examined regions in MS participants, with statistically significant reductions observed in the caudate (-25.6%, \u003cem\u003ep = 0.026\u003c/em\u003e), insula (-16.1%, \u003cem\u003ep = 0.044\u003c/em\u003e), ventral striatum (-16.8%, \u003cem\u003ep = 0.028\u003c/em\u003e), hippocampus (-18.5%, \u003cem\u003ep = 0.04\u003c/em\u003e), frontal cortex (-16.3%, \u003cem\u003ep = 0.048\u003c/em\u003e), parietal cortex (-17.1%, p = 0.026), and occipital cortex (-18.6%, \u003cem\u003ep = 0.020\u003c/em\u003e) (\u003cstrong\u003eFig. 6C\u003c/strong\u003e). In addition, we examined regional brain volumes, but no significant differences were observed between groups (\u003cstrong\u003eSupplementary Fig. 7\u003c/strong\u003e). It is important to note that these preliminary results were not corrected for multiple comparisons due to the small sample size. \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study assessed the potential of using the SV2A PET radiopharmaceutical [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 to image synaptic density loss in EAE mice at clinically relevant stages of disease progression. To our knowledge, this work provides the first evidence of tracking SV2A levels in EAE mice using a non-invasive imaging tool. Notably, the findings from the preclinical animal model have also successfully translated to human MS patients where synaptic density changes were also detectable.\u003c/p\u003e\n\u003cp\u003eSynaptic density loss is a key hallmark of MS\u003csup\u003e18,49,50\u003c/sup\u003e. Despite recent focus on studying synaptic density loss in EAE mice, no effective \u003cem\u003ein vivo\u003c/em\u003e imaging radiotracer for detecting these changes has been reported until now. In this study, we demonstrated a significant global reduction in SV2A levels in EAE mice using [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 PET imgiang, with nearly 30% loss in all analyzed regions, reinforcing the role of synaptic pathology in MS which can be captured \u003cem\u003ein vivo\u003c/em\u003e. Satisfyingly, our data is largely consistent with previously reported\u0026nbsp;\u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e values in healthy mice, showing similar \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e across brain regions with only small differences observed\u003csup\u003e51\u003c/sup\u003e. Importantly, the inflammatory environment in EAE mice may lead to modifications in perfusion dynamics and changes in cerebral blood flow, potentially resulting in altered tracer uptake\u003csup\u003e52,53\u003c/sup\u003e. This is also confirmed in our imaging study, which demonstrated a decrease in the \u003cem\u003eK\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e value of [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 uptake in EAE mice. Given the challenges in accurately quantifying SV2A levels based solely on \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u0026nbsp;\u003c/sub\u003eanalysis in EAE mice, we further evaluated tracer uptake in the target of interest using a reference region. Interestingly, we found that the amygdala displays the most stable tracer uptake profile between na\u0026iuml;ve and EAE mice, making it a suitable reference region for SUVR calculation despite its altered kinetic characteristics (\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Fig. 4\u003c/strong\u003e). Thus, since both SUVR and SRTM-2-derived DVR correlates well (\u003cstrong\u003eSupplementary\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Fig. 5\u003c/strong\u003e), we simplified the kinetic quantification analysis of [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 uptake in EAE mice by calculating SUVR, and observed a global decrease in tracer uptake in the EAE brain.\u0026nbsp;Nonetheless, postmortem autoradiography assays confirmed a significant decrease in tracer uptake in general, supporting the reliability of PET-based SV2A quantification in EAE mice. Nonetheless, [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 was specific to SV2A and blocked by LEV in EAE mice brain\u003csup\u003e54\u003c/sup\u003e. Although an insignificant decrease was observed in the hippocampus region in the ARG study, this was most likely attributed to the smaller sample size used in the experiment. The varying kinetics observed in PET imaging may also play a role in this discrepancy, as previously reported with other radiotracers\u003csup\u003e55\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs SV2A PET tracers have recently been employed as non-invasive tools to image spinal cord damage in various conditions in mice\u003csup\u003e41,56\u003c/sup\u003e, we also evaluated [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 PET imaging in the EAE spinal cord and demonstrated a significant reduction in SV2A levels in both cervical and lumbar sections. As expected, the uptake of tracers in the spinal cord regions was generally lower than in the cerebral regions, where we found a 2-3 fold difference between na\u0026iuml;ve and EAE mice, in agreement with previous reports\u003csup\u003e56,57\u003c/sup\u003e. Nonetheless, the decrease in SV2A levels in the EAE spinal cord was not as pronounced as in the brain, where we observed a 10-20% loss in the cervical and lumbar regions. Importantly, previous research has pinpointed EAE pathology in the lower sections of the spinal cord, including the lumbar region\u003csup\u003e58\u003c/sup\u003e. This area has also been successfully imaged using [\u003csup\u003e18\u003c/sup\u003eF]SynVesT-1 PET, revealing significant changes in SV2A in the lumbar section. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, we conducted SV2A PET imaging in human MS patients using the first SV2A PET radiopharmaceutical, [\u003csup\u003e11\u003c/sup\u003eC]UCB-J\u003csup\u003e33,42\u003c/sup\u003e. The primary findings indicated a widespread loss of SV2A and synaptic density, with a statistically significant 16.6% reduction in [\u003csup\u003e11\u003c/sup\u003eC]UCB-J binding in the cortical regions of MS participants compared to healthy controls. Notably, this decreasing trend in SV2A binding was observed across all analyzed brain regions, underscoring the pervasive nature of synaptic degradation in MS and the ability of SV2A PET imaging in capturing synaptic denisty loss in MS patients. Some regions, such as the caudate and frontal cortex, reached statistical significance, highlighting areas of particularly pronounced synaptic loss. Although our study did not specifically match participants by sex, previous research suggests no significant sex differences in [\u003csup\u003e11\u003c/sup\u003eC]UCB-J binding\u003csup\u003e59,60\u003c/sup\u003e. This finding supports the generalizability of our results across both sexes, particularly regarding the small cohort of this human study.\u003c/p\u003e\n\u003cp\u003eThere has been growing therapeutic effort aimed at restoring synaptic density loss in MS\u003csup\u003e8,61,62\u003c/sup\u003e. Considering the widespread reduction of synaptic density in MS, the use of a non-invasive biomarker like SV2A PET imaging, which is distributed across brain and gray matter, will offer crucial insights into studying MS and directing therapeutic strategies. Future research should investigate longitudinal changes of SV2A in EAE mice. Due to possible changes in tracer\u0026rsquo;s pharmacokinetics, conducting a model-based radiometabolite study is also advised.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eUsing PET imaging in EAE mice and human MS patients, we successfully detected significant reductions of SV2A in MS and related animal models. Our findings demonstrate that SV2A PET can reliably quantify presynaptic alterations \u003cem\u003ein vivo\u003c/em\u003e in a preclinical MS model and a clinical research study. The observed tracer uptake patterns emphasize the potential of SV2A PET for detecting diffuse synaptic pathology. Collectively, these results support the use of SV2A PET as a non-invasive imaging tool for monitoring disease progression and evaluating therapeutic interventions in MS.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cspan\u003eAnimal Ethics Statement: All animal procedures were approved by the CAMH animal Care Committee (ACC) and conducted in accordance with institutional guidelines and the relevant national regulations for the care and use of laboratory animals.\u003c/span\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStys, P. K. 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Adv.\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, eadj6187.\u003c/li\u003e\n\u003cli\u003ePoon, M. M. \u003cem\u003eet al.\u003c/em\u003e Targeting the muscarinic M1 receptor with a selective, brain-penetrant antagonist to promote remyelination in multiple sclerosis. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e \u003cstrong\u003e121\u003c/strong\u003e, e2407974121 (2024).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6769442/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6769442/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Synaptic loss is increasingly recognized as a key pathological feature in multiple sclerosis (MS), contributing to disease progression and cognitive dysfunction. Synaptic vesicle glycoprotein 2A (SV2A) positron emission tomography (PET) imaging has emerged as a promising tool for non-invasively quantifying synaptic density in vivo. Here, we evaluated the potential of SV2A PET imaging in an experimental autoimmune encephalomyelitis (EAE) mouse model and translated the findings to MS patients. In EAE mice, dynamic [18F]SynVesT-1 PET imaging revealed a significant global reduction in tracer uptake, with a nearly 30% decrease in regional distribution volume (VT) across all analyzed brain regions (p \u003c 0.0001). Correspondingly, in vitro autoradiography on rodent EAE brains corroborated the preclinical PET imaging results. We also investigated potential synaptic loss in the spinal cord of EAE mice and observed a significant decrease in SV2A levels in both the cervical and lumbar sections. In a clinical PET research study, [11C]UCB-J was used to image in MS patients (n=5) and age-matched healthy controls (n=6), and MS patients revealed a 16.6% reduction in cortical SV2A binding (p = 0.043), with significant regional reductions in the caudate (25.6%, p = 0.026), hippocampus (18.5%, p = 0.04), and frontal cortex (16.3%, p = 0.048). These findings demonstrate the application of SV2A PET imaging as a sensitive and quantitative biomarker of synaptic pathology in MS. The consistent reductions in SV2A binding observed in both preclinical and clinical research highlight the role of synaptic degeneration in MS and underscore the utility of SV2A PET imaging in MS research.","manuscriptTitle":"PET Imaging of Synaptic Density Loss in Patients with Multiple Sclerosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 13:25:33","doi":"10.21203/rs.3.rs-6769442/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"69e8b6f6-44a3-4b95-972a-649401ffc01c","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49277327,"name":"Biological sciences/Biotechnology"},{"id":49277328,"name":"Health sciences/Biomarkers/Diagnostic markers"}],"tags":[],"updatedAt":"2025-06-18T18:01:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-03 13:25:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6769442","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6769442","identity":"rs-6769442","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}