Accelerated and Localized Synucleinopathy in a Hybrid Mouse Model: Implications for Positron Emission Tomography Studies

Accelerated and Localized Synucleinopathy in a Hybrid Mouse Model: Implications for Positron Emission Tomography Studies | 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 Accelerated and Localized Synucleinopathy in a Hybrid Mouse Model: Implications for Positron Emission Tomography Studies Chunfang A Xia, Hsiu-Ming Tsai, Sandra Diaz Garcia, Shuanglong Liu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7821065/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract BACKGROUND: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by α-synuclein (α-syn) aggregation, dopamine (DA) neuron loss, and neuroinflammation. Synucleinopathy, the α-syn-related pathology, is the central to the pathogenetic processes observed in the brains of patients with PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). We are seeking an animal model with synucleinopathy that can comprehensively replicate these pathologies and adhere to suitable timeframes for preclinical research for positron emission tomography (PET) imaging studies. OBJECTIVE: To evaluate the synucleinopathy mouse model that closely replicates key pathological features through immunohistochemistry (IHC) and to assess the application of this model for PET studies. METHODS: An adeno-associated virus (AAV) carrying the mutated human α-syn gene (AAV1/2-CMV-human-A53T α-syn) and preformed fibrils (PFF) of mutated recombinant human α-syn (S87N) were co-injected into the left substantia nigra (SN) of mouse brains. PET/CT imaging and IHC were performed at different time points post-injection to detect the key pathologies in the brain. RESULTS: This model resulted in accelerated α-syn pathology, detectable as early as two weeks post-surgery, alongside DA neuron loss, microglial activation, reduced synaptic density, and impaired mitochondrial function within five weeks. The pathologies were localized, making the model suitable for PET imaging studies and/or PET ligand development. Both IHC and PET imaging confirmed the spatial relevance of these pathologies. CONCLUSIONS: This hybrid (AAV/PFF) mouse model provides an accelerated and localized platform for studying synucleinopathies such as PD, as well as for evaluating PET ligands for disease diagnosis and monitoring. Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience Parkinson’s Disease alpha-synuclein adeno-associated virus pre-formed fibrils disease model neuroinflammation dopaminergic neuron loss Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction PD is a progressive synucleinopathy pathologically characterized by the presence of α-syn aggregation, loss of nigrostriatal DA neurons, neuroinflammation, and mitochondrial dysfunction among other features 1 . As PD progresses, α-syn misfolds and aggregates, lead to the formation of Lewy bodies and Lewy neurites that spread through the brain in a prion-like manner 2 , 3 . Various studies have suggested that α-syn inclusion induced toxicity is a major factor in DA neuron death 4 . Given the complex etiology and multifactorial nature of PD, understanding the disease mechanism and identifying an ideal disease model is crucial. The ideal PD model should include the following features 5 , 6 : 1. A loss of more than 50% of the neurons, easily detectable through biochemical and neuropathological techniques; 2. The presence and development of Lewy bodies as indicators of α-syn pathology; 3. Replication of disease progression over a short period of time, facilitating faster and more cost-effective screening of potential therapeutic candidates. Rodent models of different forms of synucleinopathy have been developed using transgenic techniques, viral vector-mediated gene transferor, or inoculation with PFF seeds 7 , 8 . In transgenic mouse models, α-syn forms intracellular inclusions in many, but not all α-syn-overexpressing transgenic mice 9 . The anatomical distribution of α-syn inclusions also varies widely among models, even among those with the same promoter 10 . While these models provide some insight into protein aggregation, they do not accurately replicate the progressive loss of DA neurons seen in patients. Over the past two decades, delivering α-syn using AAV vectors has become a flexible and effective method for creating models that closely replicate the brain changes seen in PD and other Lewy body disorders 10 – 12 . But this model has limitations. The degenerative changes develop slowly, and substantial DA neuron cell loss is obtained only with very high expression levels 13 . Our previous study demonstrated that this model requires at least 4 to 5 months to develop a limited amount of α-syn aggregates (see Supplementary Fig. 1). Additionally, the inflammatory response is transient and modest magnitude. PFFs are recognized as seeds that promote the recruitment of monomeric α-syn into toxic fibrillar aggregates 14 . However, the aggregated α-syn in mice was of rodent form rather than human, and the rate of this aggregation process depends on the intracellular levels of α-syn. Thakur et al. 15 reported a combination of fibril seeds and α-syn overexpression in a rat model. The model replicates key features of human synucleinopathy, DA degeneration, impaired motor function, and inflammation. However, the model involves multiple surgeries: two AAV6-mediated α-syn vector injections into the SN and ventral tegmental area, followed by another two PFF injections four weeks later. This significantly increases the complexity of the procedure, making it challenging for researchers to replicate or standardize. Additionally, multiple surgeries cause further injury and stress to animals. Bjorklund et al. measured the combined SynFib model in rat and mouse models 16 . In their study, they observed α-syn overexpression and a loss of tyrosine hydroxylase (TH) at later time points post-injection—12 weeks in the mouse brain, 4 and 16 weeks in the rat model. PET is a powerful imaging technique used to study brain function and pathology in PD 17 . By bridging the gap between clinical symptoms and underlying pathology, PET enables earlier interventions, enhances disease management, and supports the development of targeted therapies. Currently, PET imaging for PD primarily focuses on assessing dopamine transporter activity with [ 18 F]DOPA 18 and brain metabolism with [ 18 F]FDG 19 to identify disease-specific changes. Ongoing research aims to develop additional PET ligands capable of detecting pathological α-syn, neuroinflammation, and other pathological features. An ideal animal model for PET ligand development should exhibit significant α-syn pathology that closely resembles Lewy bodies or neurites, along with neuroinflammation, and mitochondrial dysfunction, mirroring the pathologies observed in PD brains. Additionally, it should demonstrate regional and focal distributions of pathologies detectable by a PET tracer as well as temporal dynamics to allow for longitudinally studies corresponding to disease progression and ligand-binding evaluations. To address these limitations and needs, we evaluated this hybrid model that provides a more accurate, focal and comprehensive representation of PD, thereby facilitating PET ligand assessment. This model significantly accelerates α-syn pathology, observable as early as two weeks post-surgery, alongside DA neuron loss, microglial activation, reduced synaptic density, and impaired mitochondrial function at later time points. The localized injection induces focal pathologies development in mouse brain, aligning well with the requirements for effective PET ligand development for PD. Materials and Methods Animal model All animal procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Johnson & Johnson. Adult male C57BL/6J mice (Strain #000664, 10 weeks old) were purchased from Charles River (Rockville, MD). Recombinant human a-syn (S87N) purification and the in vitro generation of PFF were adapted from literatures 20 , 21 . Prior to intracranial injection, 1.5 µL of AAV1/2-CMV/CBA-human-A53T-α-syn WPRE-BGH-polyA (Charles River, 5 x10 12 gc/mL) was mixed 1:1 with sonicated recombinant human a-syn (S87N) PFF (1.5 µL, 2 mg/mL, Johnson & Johnson in Beerse, Belgium). A total of 3 µL of mixed AAV/PFF or PBS was injected unilaterally (left) into the SN at the following coordinates: anteroposterior (AP), -3.0 mm (from bregma); mediolateral (ML), -1.3 mm (from bregma); dorsoventral (DV), -4.2 mm (from dura). PET imaging was conducted at selected time points post-injection. All mice were sacrificed, and fresh-frozen brain samples were collected at 2, 3, 4, and 5 weeks post-injection for pathological analysis. Each time point included n = 3 mice for both the PBS and AAV/PFF groups. Radiosynthesis Four tracers were radiolabeled in this study. The synthesis of 18 F-labeled tracers is illustrated in Fig. 1 . [ 18 F]JNJ-CSF1R-1 22 , [ 18 F]UCB-H 23 , [ 18 F]BCPP-EF 24 , and [ 18 F]AV133 25 were synthesized as reported. The targets, radiochemical yields, specific activities, and radiochemical purities of the four tracers are presented in Supplementary Table 1. Immunohistochemistry Mouse brains were collected, immediately frozen in dry ice powder and then stored at -80ºC. The frozen mouse brains were embedded in optimal cutting temperature (OCT) compound and cut into 10 µm coronal sections that crossed the striatum or SN. To determine the expression of targets, IHC was performed. After fixing the sections in paraformaldehyde fixative solution (Alfa Aesar, MA), sections were incubated with 3% H 2 O 2 solution to quench background fluorescence. Then sections were blocked with 10% goat serum in 0.01 M PBS for 1 hour and incubated with primary antibodies overnight at 4°C. The results were visualized by fluorescent secondary antibodies (ThermoFisher, CA) under an Axio Imager 2 microscope (Carl Zeiss, Germany) and STELLARIS Confocal Microscope (Leica, IL). For signal-plex or multiplex staining, mouse brain sections were stained using TSA-Opal fluorescent detection reagents and antibodies. The staining was performed with a Leica Bond Rx autostainer (Leica, IL), followed by imaging on a Vectra Polaris™ Quantitative Pathology Imaging System (Akoya Biosciences, MA) 26 . Antibodies used in this study are listed in the Supplementary Table 2. PET/CT imaging PET/CT imaging protocols were developed based on preclinical literature 27 . An intravenous catheter was placed in each mouse prior to imaging. The animals were then positioned in the GNEXT preclinical PET/CT system (Xodus Imaging, CA, USA) on a custom 3D printed mouse-hotel bed. The ligand was administered intravenously via catheter in a volume of 150 µL of isotonic saline, followed by an additional 50 µL of fresh saline. Whole-body imaging was conducted with a field of view (FOV) measuring 120 mm × 104 mm and an average spatial resolution of less than 1 mm at the center of the FOV. List-mode data were recorded for 60 minutes for all tested tracers, followed by acquisition of a reference CT image using the embedded microCT imaging system. PET images were reconstructed using a 3D-OSEM reconstruction algorithm, which corrected for attenuation and decay, resulting in an isotropic voxel size of 540 µm. The 25 re-binned frame rate consisted of intervals: 4 × 5s, 4 × 10s, 2 × 30s, 5 × 120s, 5 × 300s, and 2 × 600s. CT images were reconstructed with a 200 µm isotropic voxel size for anatomical co-registration and attenuation correction. During imaging, the animals were maintained under 1–2% isoflurane anesthesia in oxygen, with continuous monitoring of respiration and temperature using a setup from Small Animal Instruments (SAII Inc., Stony Brook, NY, USA). Image analysis was performed using PMOD software (version 4.4, PMOD Technologies LLC), and only data collected 40–60 minutes post-injection was extracted for analysis. A three-dimensional (3D) region of interest (ROI) was defined using threshold segmentation to capture the response at the injection site in the AAV/PFF group, along with a corresponding symmetrical mirror region established as a contralateral control ROI for comparative analysis. In addition, A mouse T2-Magnetic resonance (MR) template was aligned with the PET images, facilitating visual identification of the brain regions. Results Pathological α-syn aggregation A distinguishing feature of the AAV/PFF model is the early development of inclusions and aggregates of phospho-Ser129-synuclein (p-syn) in the affected nigral DA neurons. High levels of human α-syn expression were detected in mouse brains starting at 2 weeks post AAV/PFF injection (Fig. 2 a). Human α-syn spread from the SN to most regions of the left hemisphere. The p-syn was found to be more concentrated around the SN compared to total α-syn (Fig. 2 a). Double staining showed colocalization of both markers (Fig. 2 b). Lewy body-like p-syn pathology was observed in many neurons around SN region (indicated by arrows), as well as in distorted axons and dendrites, resembling Lewy neurites (marked by arrowheads in Fig. 2 b). Dopaminergic neuron loss Representative double staining for vesicular monoamine transporter 2 (VMAT2) and tyrosine hydroxylase (TH) revealed significant DA neuron loss in the striatum as shown in Fig. 3 a. The intensity of TH, measured by IHC in the striatum of injection side, demonstrated a gradual reduction over time post the injection (Fig. 3 b). Specifically, TH intensity was measured at 79%, 77%, 65%, and 59% of contralateral side at 2, 3, 4, and 5 weeks post-injection, respectively (Fig. 3 b, n = 3). PET/CT scan was conducted at 4 weeks post-injection. The axial and coronal PET images illustrated the patterns of brain uptake in both PBS and AAV/PFF groups using a VMAT2 tracer, [ 18 F]AV-133 (Fig. 3 c). The uptake in the ipsilateral and contralateral striatum did not show significant differences with PBS injection. In contrast, the AAV/PFF mice displayed a decreased uptake on the injection side 4 weeks post injection (Fig. 3 c). A ∼40% reduction in uptake was observed in the ipsilateral striatum compared to the contralateral side, as illustrated in Fig. 3 d. Neuroinflammation A microglial reaction was detected by Iba1 staining spreading from the left SN to the midbrain and thalamus, from 2 to 5 weeks post-injection (Fig. 4 a). Multiplex staining of p-syn, Iba1, and triggering receptor expressed on myeloid cells 2 (TREM2), provided clear evidence of inflammatory responses localized to regions with pathological p-syn deposits at 3 weeks post-surgery (Fig. 4 b). The results showed that the inflammatory response expanded in correlation with the presence of pathological α-syn, demonstrating pronounced microglial activation in the affected areas. This sharply contrasts with the contralateral side of the brain, where such inflammatory markers were notably less prominent, and cellular morphology indicated non-reactive microglia (Fig. 4 c). Colony-stimulating factor 1 receptor (CSF1R), a marker of neuroinflammation primarily expressed by microglia in the central nervous system, exhibited increased expression in the presence of p-syn following 3 weeks of AAV/PFF treatment, as illustrated in Fig. 5 a. The small molecule PET ligand [ 18 F]JNJ-CSF1R-1 was used to target CSF1R and detect neuroinflammation 22 , 28 . MR template alignment with the PET images allowed for accurate localization of the SN area, correlating well with IHC results obtained from brain tissue cryosections. CSF1R PET imaging revealed elevated uptake in the left SN, indicating increased inflammation around the injection site. The uptake patterns identified in PET imaging were in good agreement with the IHC findings (Fig. 5 b and 5 c). The statistical analysis of the PET images indicated that [ 18 F]JNJ-CSF1R-1 uptake at the injection site was 42% increased comparing to that at the contralateral site (Fig. 5 d, n = 3), which aligned with the statistical findings from the IHC analysis (Fig. 5 e, n = 3). Synapse Integrity Synaptic density is measured by synaptic vesicle protein 2A (SV2A), which is considered a key marker for synaptic density. SV2A IHC revealed a significant reduction in synaptic density around left SN area (Fig. 6 a) at 5 weeks post injection. Compared to the contralateral side of the brain, where synaptic integrity remained relatively intact (Fig. 6 b). The PET images illustrate synaptic density patterns of brain uptake in both the PBS and AAV/PFF groups using the [ 18 F]UCB-H tracer, employing axial and coronal sections in Fig. 6 c. Identical ROIs were applied to both the PBS and AAV/PFF groups for evaluation. The uptake in the ipsilateral and contralateral sides did not show significant differences in PBS group. In the AAV/PFF-treated group, an 18% reduction in tracer uptake was observed in the left SN compared to the right side (n = 3, P = 0.068), indicating a significant change in synaptic density around the injection site (Fig. 6 c). Mitochondrial dysfunction IHC of ATP synthase subunit A (ATP5A), a key mitochondrial marker, revealed a significant decrease in expression correlated with the extent of α-syn pathology observed at 5 weeks post-injection comparing to the contralateral side (Fig. 7 a and 7 b). The PET imaging of [ 18 F] BCPP-EF tracer was used to analyze mitochondrial integrity in both the PBS and AAV/PFF groups (shown in Fig. 7 c). The brain uptake in the ipsilateral and contralateral SN showed no significant differences in the PBS group, indicating preserved mitochondrial integrity. In contrast, the AAV/PFF group demonstrated a slight but significant reduction of 7.3% in uptake between the left and right SN (n = 3, *P < 0.01 ) , as depicted in Fig. 7 c. Statistical Analysis All statistical analyses were conducted using GraphPad Prism software version 9 (Prism, CA, USA). Data are presented as the mean ± standard deviation (SD). An ordinary one-way ANOVA with multiple comparisons was employed to compare three or more groups, while comparisons between two groups were performed using a t-test. A P-value of < 0.05 indicates statistical significance. Discussion Rodent models of synucleinopathy, such as PFF injections 29 or AAV-mediated α-syn overexpression (Supplementary Fig. 1), develop pathology slowly and show only minimal changes in key features over time. In contrast, the hybrid AAV/PFF model introduced in this study is specifically designed to address these limitations, making it exceptionally suitable for PET imaging applications. Our results demonstrate that this hybrid model induces accelerated and significant amount of human toxic α-syn deposits, which become detectable as early as two weeks post-surgery. The model effectively combines key features of the α-syn PFF and AAV-α-syn models, including the generation of toxic α-syn aggregates and Lewy body- and Lewy neurite-like inclusions induced by the PFF seeds, along with increased cellular levels of human α-syn driven by the AAV vector. Consequently, in response to pathological α-syn aggregation, the acute toxicity and dopaminergic neuron death were detected by TH and VMAT2 IHC. The DA neuron intensity in striatum is progressively reduced from 21% to 41% from 2 to 5 weeks post injection. Neuroinflammation is recognized as a key factor in PD, which contributes to neurodegeneration by accelerating brain aging and inhibiting regeneration 30 . In this model, the increased microglial activation, assessed using Iba1, TREM2, and CSF1R staining, indicates a significant inflammatory response. This response developed rapidly and shows regional specificity, being most prominently expressed in areas with pathological α-syn inclusions. SV2A, a synaptic protein, plays a vital role in the regulation of neurotransmitter release and is commonly used as a marker for synaptic density 31 . Notably, synaptic deficits are detected at later time point (5 weeks) in this model. The loss of synaptic density is most pronounced in regions exhibiting pathological hallmarks, further supporting the idea that synaptic impairment is a key feature of dopaminergic neurodegeneration. Additionally, mitochondrial dysfunction is closely associated with genetic forms of PD, with several PARK genes being directly linked to impaired mitochondrial function and integrity 32 . ATP5A staining revealed substantial mitochondrial dysfunction in the same region where pathological α-syn is present. The interplay between synaptic deficits and mitochondrial dysfunction underscores the comprehensive nature of this model in capturing the multifaceted aspects of PD pathology. The focal and accelerated pathology development of this model also provides an ideal platform for PET ligand validation. In this study, several PET ligands were employed to examine various aspects of the disease. The [ 18 F]AV-133 33 PET ligand, which targets VMAT2, detected approximately 40% loss of dopamine in the left striatum at 4 weeks post-injection, consistent with the levels of TH and VMAT2 protein measured by IHC. Enhanced neuroinflammation in this model was detected using [ 18 F]JNJ-CSF1R-1 22, 28 PET imaging, demonstrating a similar pattern of positive signals as visualized by CSF1R IHC. The [ 18 F]UCB-H 34 tracer, developed as a small molecule PET ligand targeting SV2A, a marker of synaptic density, revealed an 18% reduction in synaptic integrity around injection site. The use of [ 18 F]UCB-H allowed for the visualization and quantification of these changes in vivo. Additionally, [ 18 F]BCPP-EF, which targets mitochondrial complex I (MC-1), offered important insights into mitochondrial dysfunction in PD, showing a small but statistically significant decrease of 7.3% in mitochondrial integrity. This finding underscores the impact of PD on mitochondrial function In conclusion, this accelerated and localized synucleinopathy mouse model provides a robust and reliable platform for studying PD pathologies and assessing potential PET ligands. Its capacity to replicate key PD features in a short timeframe makes it an invaluable tool for preclinical research and drug development. Future studies should focus on optimizing AAV/PFF dosing, incorporating additional time points to strengthen model validation, and exploring its applications in evaluating novel PET ligands and PD therapies. Declarations Competing interests: All authors declare no financial or non-financial competing interests. Funding statement: This study was fully funded by Johnson & Johnson. Author Contribution Author contributions: C Xia : conceptualization, formal analysis, investigation, methodology, project administration, visualization, writing-original draft, writing-review & editing. HM Tsai: formal analysis, investigation, writing-original draft. SD Garcia: formal analysis, investigation. SL Liu: investigation. A Matzeu : investigation. M Salarian : methodology. W Bruinzeel : investigation. AK Szardenings : conceptualization, writing-review & editing, and supervision. 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Additional Declarations No competing interests reported. Supplementary Files HybridsynucleinopathymousemodelSupplementaryMaterials102025Xia.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 20 Nov, 2025 Reviews received at journal 19 Nov, 2025 Reviews received at journal 09 Nov, 2025 Reviewers agreed at journal 03 Nov, 2025 Reviewers agreed at journal 18 Oct, 2025 Reviewers invited by journal 16 Oct, 2025 Editor assigned by journal 16 Oct, 2025 Submission checks completed at journal 13 Oct, 2025 First submitted to journal 09 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7821065","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":537062838,"identity":"dd0d2633-f2a5-4e08-ba26-d4cc923e2abe","order_by":0,"name":"Chunfang A 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06:49:04","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97910,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/133211347c25c86cf9b30b76.html"},{"id":94760910,"identity":"2e126917-9bda-4c62-8abb-6d8faf687a98","added_by":"auto","created_at":"2025-10-30 12:06:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eRadiolabeling reactions for [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]JNJ-CSF1R-1 (A), [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]UCB-H (B), [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]BCPP-EF (C), and [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]AV133 (D).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/9508ea27c5921ad1080c48fb.png"},{"id":94760916,"identity":"09950a64-505f-4b2e-ab07-c7cfd8c894fc","added_by":"auto","created_at":"2025-10-30 12:06:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1485426,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHuman α-syn and p-syn expression in mouse brains from 2 to 5 weeks post-injection. (a) Human α-syn monomer and p-syn IHC on coronal brain sections across SN. Yellow stars indicate the injection site. \u0026nbsp;(b) Double immunostaining of α-syn and p-syn on mouse brain sections at 4 weeks post-injection. P-syn positive staining was more focal around the SN compared to α-syn monomer expression. Lewy body-like (arrows) and Lewy neurites-like (arrow heads) structures can be found under confocal microscope (b).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/0dfc16532cd0d82d4a88336f.png"},{"id":94823407,"identity":"792115db-c0f1-4f84-ba4a-b16ad1c63d37","added_by":"auto","created_at":"2025-10-31 06:47:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":314383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDA neuron degradation in the striatum was detected by IHC and PET imaging. (a) Representative images of double staining of VMAT2 and TH on mouse brain sections across the striatum at 4 weeks post AAV/PFF injection. (b) DA neuron deficiency in the striatum was observed at all time points from 2 to 5 weeks (n=3). (c) Comparison of VMAT2 PET imaging in the mouse brain at 4 weeks post-injection between PBS and AAV/PFF. (d) Percentage of uptake of contralateral sides (n=3, *P\u0026lt;0.005).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/392cb71d0f86be19f2121b7d.png"},{"id":94760913,"identity":"41732fbf-b640-4551-9231-941d5b81542a","added_by":"auto","created_at":"2025-10-30 12:06:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1032393,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eNeuroinflammation was detected colocalized with aggregated α-syn deposits. (a) Iba1 staining on mouse brain sections from 2 to 5 weeks post AAV/PFF injection. (b) Multiplex staining of p-syn, Iba1, and TREM2 IHC on mouse brain tissue collected 3 weeks post AAV/PFF injection. (c) Increased Iba1 and TREM2 protein expression levels were observed in regions with overexpressed p-syn.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/54e5dc5837e3fdfbff041039.png"},{"id":94760923,"identity":"70482b00-d508-463a-8068-3b4f29e61e1e","added_by":"auto","created_at":"2025-10-30 12:06:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":679781,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCSF1R expression detected by IHC and PET imaging around the left SN at 3 weeks post-surgery. (a) IHC indicates that CSF1R levels increased with the presence of p-syn (scale bar = 50 µm). (b, c) In vivo PET signal of [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]JNJ-CSF1R-1 and CSF1R IHC was compared side-by-side in same animal. The bar charts (d and e) illustrated the increased signal in injection side comparing to contralateral side based on IHC and PET images (n=3, *P\u0026lt;0.01).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/efbbc2f65cab3103cc7ced20.png"},{"id":94824588,"identity":"27c470ec-5da7-44b2-a13f-f207e53beff2","added_by":"auto","created_at":"2025-10-31 06:49:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":938058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eSV2A density reduction detected by SV2A IHC and PET imaging using [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]UCB-H. (a, b) SV2A IHC on brain sections across the SN. (c) [\u003c/em\u003e\u003csup\u003e\u003cem\u003e18\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eF]UCB-H PET tracer images and statistical analysis of left SN (indicated by the blue arrow) for both PBS and AAV/PFF (n=3, *P=0.068).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/633319d8eb60537cc7b5fbcb.png"},{"id":94824152,"identity":"f4850d9e-d233-4fea-a64e-f6628c720647","added_by":"auto","created_at":"2025-10-31 06:48:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":823559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eMitochondrial dysfunction detected by ATP5A IHC (a, b) and MC-1 PET tracer images, along with statistical analysis of the injection site (indicated by the blue arrow) from PBS and AAV/PFF (n=3, *P\u0026lt;\u003c/em\u003e0.01\u003cem\u003e) (c).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/27260079a19a9e43fe429102.png"},{"id":94827257,"identity":"6b73868f-c7f4-4e05-a220-54aa84bf699e","added_by":"auto","created_at":"2025-10-31 06:56:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5681922,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/8a51ffb7-2c2f-4677-81f3-97daff4e17cb.pdf"},{"id":94824485,"identity":"0faad549-c22e-4906-a415-f73de5d4bd91","added_by":"auto","created_at":"2025-10-31 06:49:02","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":555146,"visible":true,"origin":"","legend":"","description":"","filename":"HybridsynucleinopathymousemodelSupplementaryMaterials102025Xia.docx","url":"https://assets-eu.researchsquare.com/files/rs-7821065/v1/4e37ebb55bbddccf42b3d79c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Accelerated and Localized Synucleinopathy in a Hybrid Mouse Model: Implications for Positron Emission Tomography Studies","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePD is a progressive synucleinopathy pathologically characterized by the presence of α-syn aggregation, loss of nigrostriatal DA neurons, neuroinflammation, and mitochondrial dysfunction among other features\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As PD progresses, α-syn misfolds and aggregates, lead to the formation of Lewy bodies and Lewy neurites that spread through the brain in a prion-like manner\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Various studies have suggested that α-syn inclusion induced toxicity is a major factor in DA neuron death\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Given the complex etiology and multifactorial nature of PD, understanding the disease mechanism and identifying an ideal disease model is crucial. The ideal PD model should include the following features \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e: 1. A loss of more than 50% of the neurons, easily detectable through biochemical and neuropathological techniques; 2. The presence and development of Lewy bodies as indicators of α-syn pathology; 3. Replication of disease progression over a short period of time, facilitating faster and more cost-effective screening of potential therapeutic candidates.\u003c/p\u003e\u003cp\u003eRodent models of different forms of synucleinopathy have been developed using transgenic techniques, viral vector-mediated gene transferor, or inoculation with PFF seeds\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In transgenic mouse models, α-syn forms intracellular inclusions in many, but not all α-syn-overexpressing transgenic mice\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The anatomical distribution of α-syn inclusions also varies widely among models, even among those with the same promoter\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While these models provide some insight into protein aggregation, they do not accurately replicate the progressive loss of DA neurons seen in patients. Over the past two decades, delivering α-syn using AAV vectors has become a flexible and effective method for creating models that closely replicate the brain changes seen in PD and other Lewy body disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. But this model has limitations. The degenerative changes develop slowly, and substantial DA neuron cell loss is obtained only with very high expression levels\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Our previous study demonstrated that this model requires at least 4 to 5 months to develop a limited amount of α-syn aggregates (see Supplementary Fig.\u0026nbsp;1). Additionally, the inflammatory response is transient and modest magnitude. PFFs are recognized as seeds that promote the recruitment of monomeric α-syn into toxic fibrillar aggregates\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, the aggregated α-syn in mice was of rodent form rather than human, and the rate of this aggregation process depends on the intracellular levels of α-syn. Thakur et al.\u003csup\u003e15\u003c/sup\u003e reported a combination of fibril seeds and α-syn overexpression in a rat model. The model replicates key features of human synucleinopathy, DA degeneration, impaired motor function, and inflammation. However, the model involves multiple surgeries: two AAV6-mediated α-syn vector injections into the SN and ventral tegmental area, followed by another two PFF injections four weeks later. This significantly increases the complexity of the procedure, making it challenging for researchers to replicate or standardize. Additionally, multiple surgeries cause further injury and stress to animals. Bjorklund et al. measured the combined SynFib model in rat and mouse models\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In their study, they observed α-syn overexpression and a loss of tyrosine hydroxylase (TH) at later time points post-injection\u0026mdash;12 weeks in the mouse brain, 4 and 16 weeks in the rat model.\u003c/p\u003e\u003cp\u003ePET is a powerful imaging technique used to study brain function and pathology in PD\u003csup\u003e17\u003c/sup\u003e. By bridging the gap between clinical symptoms and underlying pathology, PET enables earlier interventions, enhances disease management, and supports the development of targeted therapies. Currently, PET imaging for PD primarily focuses on assessing dopamine transporter activity with [\u003csup\u003e18\u003c/sup\u003eF]DOPA\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and brain metabolism with [\u003csup\u003e18\u003c/sup\u003eF]FDG\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e to identify disease-specific changes. Ongoing research aims to develop additional PET ligands capable of detecting pathological α-syn, neuroinflammation, and other pathological features. An ideal animal model for PET ligand development should exhibit significant α-syn pathology that closely resembles Lewy bodies or neurites, along with neuroinflammation, and mitochondrial dysfunction, mirroring the pathologies observed in PD brains. Additionally, it should demonstrate regional and focal distributions of pathologies detectable by a PET tracer as well as temporal dynamics to allow for longitudinally studies corresponding to disease progression and ligand-binding evaluations.\u003c/p\u003e\u003cp\u003eTo address these limitations and needs, we evaluated this hybrid model that provides a more accurate, focal and comprehensive representation of PD, thereby facilitating PET ligand assessment. This model significantly accelerates α-syn pathology, observable as early as two weeks post-surgery, alongside DA neuron loss, microglial activation, reduced synaptic density, and impaired mitochondrial function at later time points. The localized injection induces focal pathologies development in mouse brain, aligning well with the requirements for effective PET ligand development for PD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eAnimal model\u003c/p\u003e\u003cp\u003eAll animal procedures adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Johnson \u0026amp; Johnson. Adult male C57BL/6J mice (Strain #000664, 10 weeks old) were purchased from Charles River (Rockville, MD). Recombinant human a-syn (S87N) purification and the in vitro generation of PFF were adapted from literatures \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Prior to intracranial injection, 1.5 \u0026micro;L of AAV1/2-CMV/CBA-human-A53T-α-syn WPRE-BGH-polyA (Charles River, 5 x10\u003csup\u003e12\u003c/sup\u003e gc/mL) was mixed 1:1 with sonicated recombinant human a-syn (S87N) PFF (1.5 \u0026micro;L, 2 mg/mL, Johnson \u0026amp; Johnson in Beerse, Belgium). A total of 3 \u0026micro;L of mixed AAV/PFF or PBS was injected unilaterally (left) into the SN at the following coordinates: anteroposterior (AP), -3.0 mm (from bregma); mediolateral (ML), -1.3 mm (from bregma); dorsoventral (DV), -4.2 mm (from dura). PET imaging was conducted at selected time points post-injection. All mice were sacrificed, and fresh-frozen brain samples were collected at 2, 3, 4, and 5 weeks post-injection for pathological analysis. Each time point included n\u0026thinsp;=\u0026thinsp;3 mice for both the PBS and AAV/PFF groups.\u003c/p\u003e\u003cp\u003eRadiosynthesis\u003c/p\u003e\u003cp\u003eFour tracers were radiolabeled in this study. The synthesis of \u003csup\u003e18\u003c/sup\u003eF-labeled tracers is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. [\u003csup\u003e18\u003c/sup\u003eF]JNJ-CSF1R-1\u003csup\u003e22\u003c/sup\u003e, [\u003csup\u003e18\u003c/sup\u003eF]UCB-H\u003csup\u003e23\u003c/sup\u003e, [\u003csup\u003e18\u003c/sup\u003eF]BCPP-EF\u003csup\u003e24\u003c/sup\u003e, and [\u003csup\u003e18\u003c/sup\u003eF]AV133\u003csup\u003e25\u003c/sup\u003e were synthesized as reported. The targets, radiochemical yields, specific activities, and radiochemical purities of the four tracers are presented in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eImmunohistochemistry\u003c/p\u003e\u003cp\u003eMouse brains were collected, immediately frozen in dry ice powder and then stored at -80\u0026ordm;C. The frozen mouse brains were embedded in optimal cutting temperature (OCT) compound and cut into 10 \u0026micro;m coronal sections that crossed the striatum or SN. To determine the expression of targets, IHC was performed. After fixing the sections in paraformaldehyde fixative solution (Alfa Aesar, MA), sections were incubated with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution to quench background fluorescence. Then sections were blocked with 10% goat serum in 0.01 M PBS for 1 hour and incubated with primary antibodies overnight at 4\u0026deg;C. The results were visualized by fluorescent secondary antibodies (ThermoFisher, CA) under an Axio Imager 2 microscope (Carl Zeiss, Germany) and STELLARIS Confocal Microscope (Leica, IL).\u003c/p\u003e\u003cp\u003eFor signal-plex or multiplex staining, mouse brain sections were stained using TSA-Opal fluorescent detection reagents and antibodies. The staining was performed with a Leica Bond Rx autostainer (Leica, IL), followed by imaging on a Vectra Polaris\u0026trade; Quantitative Pathology Imaging System (Akoya Biosciences, MA)\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Antibodies used in this study are listed in the Supplementary Table\u0026nbsp;2.\u003c/p\u003e\u003cp\u003ePET/CT imaging\u003c/p\u003e\u003cp\u003ePET/CT imaging protocols were developed based on preclinical literature\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. An intravenous catheter was placed in each mouse prior to imaging. The animals were then positioned in the GNEXT preclinical PET/CT system (Xodus Imaging, CA, USA) on a custom 3D printed mouse-hotel bed. The ligand was administered intravenously via catheter in a volume of 150 \u0026micro;L of isotonic saline, followed by an additional 50 \u0026micro;L of fresh saline. Whole-body imaging was conducted with a field of view (FOV) measuring 120 mm \u0026times; 104 mm and an average spatial resolution of less than 1 mm at the center of the FOV. List-mode data were recorded for 60 minutes for all tested tracers, followed by acquisition of a reference CT image using the embedded microCT imaging system. PET images were reconstructed using a 3D-OSEM reconstruction algorithm, which corrected for attenuation and decay, resulting in an isotropic voxel size of 540 \u0026micro;m. The 25 re-binned frame rate consisted of intervals: 4 \u0026times; 5s, 4 \u0026times; 10s, 2 \u0026times; 30s, 5 \u0026times; 120s, 5 \u0026times; 300s, and 2 \u0026times; 600s. CT images were reconstructed with a 200 \u0026micro;m isotropic voxel size for anatomical co-registration and attenuation correction. During imaging, the animals were maintained under 1\u0026ndash;2% isoflurane anesthesia in oxygen, with continuous monitoring of respiration and temperature using a setup from Small Animal Instruments (SAII Inc., Stony Brook, NY, USA). Image analysis was performed using PMOD software (version 4.4, PMOD Technologies LLC), and only data collected 40\u0026ndash;60 minutes post-injection was extracted for analysis. A three-dimensional (3D) region of interest (ROI) was defined using threshold segmentation to capture the response at the injection site in the AAV/PFF group, along with a corresponding symmetrical mirror region established as a contralateral control ROI for comparative analysis. In addition, A mouse T2-Magnetic resonance (MR) template was aligned with the PET images, facilitating visual identification of the brain regions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003ePathological α-syn aggregation\u003c/p\u003e\u003cp\u003eA distinguishing feature of the AAV/PFF model is the early development of inclusions and aggregates of phospho-Ser129-synuclein (p-syn) in the affected nigral DA neurons. High levels of human α-syn expression were detected in mouse brains starting at 2 weeks post AAV/PFF injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Human α-syn spread from the SN to most regions of the left hemisphere. The p-syn was found to be more concentrated around the SN compared to total α-syn (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Double staining showed colocalization of both markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Lewy body-like p-syn pathology was observed in many neurons around SN region (indicated by arrows), as well as in distorted axons and dendrites, resembling Lewy neurites (marked by arrowheads in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eDopaminergic neuron loss\u003c/p\u003e\u003cp\u003eRepresentative double staining for vesicular monoamine transporter 2 (VMAT2) and tyrosine hydroxylase (TH) revealed significant DA neuron loss in the striatum as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. The intensity of TH, measured by IHC in the striatum of injection side, demonstrated a gradual reduction over time post the injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Specifically, TH intensity was measured at 79%, 77%, 65%, and 59% of contralateral side at 2, 3, 4, and 5 weeks post-injection, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, n\u0026thinsp;=\u0026thinsp;3). PET/CT scan was conducted at 4 weeks post-injection. The axial and coronal PET images illustrated the patterns of brain uptake in both PBS and AAV/PFF groups using a VMAT2 tracer, [\u003csup\u003e18\u003c/sup\u003eF]AV-133 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The uptake in the ipsilateral and contralateral striatum did not show significant differences with PBS injection. In contrast, the AAV/PFF mice displayed a decreased uptake on the injection side 4 weeks post injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). A \u0026sim;40% reduction in uptake was observed in the ipsilateral striatum compared to the contralateral side, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNeuroinflammation\u003c/p\u003e\u003cp\u003eA microglial reaction was detected by Iba1 staining spreading from the left SN to the midbrain and thalamus, from 2 to 5 weeks post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Multiplex staining of p-syn, Iba1, and triggering receptor expressed on myeloid cells 2 (TREM2), provided clear evidence of inflammatory responses localized to regions with pathological p-syn deposits at 3 weeks post-surgery (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The results showed that the inflammatory response expanded in correlation with the presence of pathological α-syn, demonstrating pronounced microglial activation in the affected areas. This sharply contrasts with the contralateral side of the brain, where such inflammatory markers were notably less prominent, and cellular morphology indicated non-reactive microglia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eColony-stimulating factor 1 receptor (CSF1R), a marker of neuroinflammation primarily expressed by microglia in the central nervous system, exhibited increased expression in the presence of p-syn following 3 weeks of AAV/PFF treatment, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. The small molecule PET ligand [\u003csup\u003e18\u003c/sup\u003eF]JNJ-CSF1R-1 was used to target CSF1R and detect neuroinflammation\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. MR template alignment with the PET images allowed for accurate localization of the SN area, correlating well with IHC results obtained from brain tissue cryosections. CSF1R PET imaging revealed elevated uptake in the left SN, indicating increased inflammation around the injection site. The uptake patterns identified in PET imaging were in good agreement with the IHC findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The statistical analysis of the PET images indicated that [\u003csup\u003e18\u003c/sup\u003eF]JNJ-CSF1R-1 uptake at the injection site was 42% increased comparing to that at the contralateral site (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, n\u0026thinsp;=\u0026thinsp;3), which aligned with the statistical findings from the IHC analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSynapse Integrity\u003c/p\u003e\u003cp\u003eSynaptic density is measured by synaptic vesicle protein 2A (SV2A), which is considered a key marker for synaptic density. SV2A IHC revealed a significant reduction in synaptic density around left SN area (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) at 5 weeks post injection. Compared to the contralateral side of the brain, where synaptic integrity remained relatively intact (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The PET images illustrate synaptic density patterns of brain uptake in both the PBS and AAV/PFF groups using the [\u003csup\u003e18\u003c/sup\u003eF]UCB-H tracer, employing axial and coronal sections in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec. Identical ROIs were applied to both the PBS and AAV/PFF groups for evaluation. The uptake in the ipsilateral and contralateral sides did not show significant differences in PBS group. In the AAV/PFF-treated group, an 18% reduction in tracer uptake was observed in the left SN compared to the right side (n\u0026thinsp;=\u0026thinsp;3, P\u0026thinsp;=\u0026thinsp;0.068), indicating a significant change in synaptic density around the injection site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMitochondrial dysfunction\u003c/p\u003e\u003cp\u003eIHC of ATP synthase subunit A (ATP5A), a key mitochondrial marker, revealed a significant decrease in expression correlated with the extent of α-syn pathology observed at 5 weeks post-injection comparing to the contralateral side (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). The PET imaging of [\u003csup\u003e18\u003c/sup\u003eF] BCPP-EF tracer was used to analyze mitochondrial integrity in both the PBS and AAV/PFF groups (shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). The brain uptake in the ipsilateral and contralateral SN showed no significant differences in the PBS group, indicating preserved mitochondrial integrity. In contrast, the AAV/PFF group demonstrated a slight but significant reduction of 7.3% in uptake between the left and right SN (n\u0026thinsp;=\u0026thinsp;3, \u003cem\u003e*P\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003cem\u003e)\u003c/em\u003e, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analyses were conducted using GraphPad Prism software version 9 (Prism, CA, USA). Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). An ordinary one-way ANOVA with multiple comparisons was employed to compare three or more groups, while comparisons between two groups were performed using a t-test. A P-value of \u0026lt;\u0026thinsp;0.05 indicates statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRodent models of synucleinopathy, such as PFF injections\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e or AAV-mediated α-syn overexpression (Supplementary Fig.\u0026nbsp;1), develop pathology slowly and show only minimal changes in key features over time. In contrast, the hybrid AAV/PFF model introduced in this study is specifically designed to address these limitations, making it exceptionally suitable for PET imaging applications.\u003c/p\u003e\u003cp\u003eOur results demonstrate that this hybrid model induces accelerated and significant amount of human toxic α-syn deposits, which become detectable as early as two weeks post-surgery. The model effectively combines key features of the α-syn PFF and AAV-α-syn models, including the generation of toxic α-syn aggregates and Lewy body- and Lewy neurite-like inclusions induced by the PFF seeds, along with increased cellular levels of human α-syn driven by the AAV vector. Consequently, in response to pathological α-syn aggregation, the acute toxicity and dopaminergic neuron death were detected by TH and VMAT2 IHC. The DA neuron intensity in striatum is progressively reduced from 21% to 41% from 2 to 5 weeks post injection. Neuroinflammation is recognized as a key factor in PD, which contributes to neurodegeneration by accelerating brain aging and inhibiting regeneration\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this model, the increased microglial activation, assessed using Iba1, TREM2, and CSF1R staining, indicates a significant inflammatory response. This response developed rapidly and shows regional specificity, being most prominently expressed in areas with pathological α-syn inclusions. SV2A, a synaptic protein, plays a vital role in the regulation of neurotransmitter release and is commonly used as a marker for synaptic density\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Notably, synaptic deficits are detected at later time point (5 weeks) in this model. The loss of synaptic density is most pronounced in regions exhibiting pathological hallmarks, further supporting the idea that synaptic impairment is a key feature of dopaminergic neurodegeneration. Additionally, mitochondrial dysfunction is closely associated with genetic forms of PD, with several \u003cem\u003ePARK\u003c/em\u003e genes being directly linked to impaired mitochondrial function and integrity\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. ATP5A staining revealed substantial mitochondrial dysfunction in the same region where pathological α-syn is present. The interplay between synaptic deficits and mitochondrial dysfunction underscores the comprehensive nature of this model in capturing the multifaceted aspects of PD pathology.\u003c/p\u003e\u003cp\u003eThe focal and accelerated pathology development of this model also provides an ideal platform for PET ligand validation. In this study, several PET ligands were employed to examine various aspects of the disease. The [\u003csup\u003e18\u003c/sup\u003eF]AV-133\u003csup\u003e33\u003c/sup\u003e PET ligand, which targets VMAT2, detected approximately 40% loss of dopamine in the left striatum at 4 weeks post-injection, consistent with the levels of TH and VMAT2 protein measured by IHC. Enhanced neuroinflammation in this model was detected using [\u003csup\u003e18\u003c/sup\u003eF]JNJ-CSF1R-1\u003csup\u003e22, 28\u003c/sup\u003e PET imaging, demonstrating a similar pattern of positive signals as visualized by CSF1R IHC. The [\u003csup\u003e18\u003c/sup\u003eF]UCB-H \u003csup\u003e34\u003c/sup\u003e tracer, developed as a small molecule PET ligand targeting SV2A, a marker of synaptic density, revealed an 18% reduction in synaptic integrity around injection site. The use of [\u003csup\u003e18\u003c/sup\u003eF]UCB-H allowed for the visualization and quantification of these changes in vivo. Additionally, [\u003csup\u003e18\u003c/sup\u003eF]BCPP-EF, which targets mitochondrial complex I (MC-1), offered important insights into mitochondrial dysfunction in PD, showing a small but statistically significant decrease of 7.3% in mitochondrial integrity. This finding underscores the impact of PD on mitochondrial function\u003c/p\u003e\u003cp\u003eIn conclusion, this accelerated and localized synucleinopathy mouse model provides a robust and reliable platform for studying PD pathologies and assessing potential PET ligands. Its capacity to replicate key PD features in a short timeframe makes it an invaluable tool for preclinical research and drug development. Future studies should focus on optimizing AAV/PFF dosing, incorporating additional time points to strengthen model validation, and exploring its applications in evaluating novel PET ligands and PD therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests:\u003c/h2\u003e\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding statement:\u003c/h2\u003e\u003cp\u003eThis study was fully funded by Johnson \u0026amp; Johnson.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor contributions: C Xia : conceptualization, formal analysis, investigation, methodology, project administration, visualization, writing-original draft, writing-review \u0026amp; editing. HM Tsai: formal analysis, investigation, writing-original draft. SD Garcia: formal analysis, investigation. SL Liu: investigation. A Matzeu : investigation. M Salarian : methodology. W Bruinzeel : investigation. AK Szardenings : conceptualization, writing-review \u0026amp; editing, and supervision.\u003c/p\u003e\u003ch2\u003eData Availability:\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDickson DW. Parkinson's disease and parkinsonism: neuropathology. \u003cem\u003eCold Spring Harb Perspect Med\u003c/em\u003e 2012; 2 2012/08/22. DOI: 10.1101/cshperspect.a009258.\u003c/li\u003e\n\u003cli\u003eVisanji NP, Brotchie JM, Kalia LV, et al. alpha-Synuclein-Based Animal Models of Parkinson's Disease: Challenges and Opportunities in a New Era. \u003cem\u003eTrends Neurosci\u003c/em\u003e 2016; 39: 750-762. 2016/10/26. DOI: 10.1016/j.tins.2016.09.003.\u003c/li\u003e\n\u003cli\u003eNegi S, Khurana N and Duggal N. 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Characteristics of the 18F-fluorodeoxyglucose ((18)F-FDG) and [(18)F] 9-fluoropropyl-(+)-dihydrotetrabenazine ((18)F-FP-DTBZ) positron emission tomography (PET) in patients with cognitive impairment in Parkinson's disease. \u003cem\u003eClin Radiol\u003c/em\u003e 2025; 89: 107038. 2025/09/04. DOI: 10.1016/j.crad.2025.107038.\u003c/li\u003e\n\u003cli\u003eGiasson BI, Murray IV, Trojanowski JQ, et al. A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly. \u003cem\u003eJ Biol Chem\u003c/em\u003e 2001; 276: 2380-2386. 2000/11/04. DOI: 10.1074/jbc.M008919200.\u003c/li\u003e\n\u003cli\u003eLuk KC, Song C, O'Brien P, et al. Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 2009; 106: 20051-20056. 2009/11/07. DOI: 10.1073/pnas.0908005106.\u003c/li\u003e\n\u003cli\u003eSalarian M, Liu S, Tsai HM, et al. 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DOI: 10.2967/jnumed.113.136143.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Parkinson’s Disease, alpha-synuclein, adeno-associated virus, pre-formed fibrils, disease model, neuroinflammation, dopaminergic neuron loss","lastPublishedDoi":"10.21203/rs.3.rs-7821065/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7821065/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBACKGROUND:\u003c/h2\u003e\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a neurodegenerative disorder characterized by α-synuclein (α-syn) aggregation, dopamine (DA) neuron loss, and neuroinflammation. Synucleinopathy, the α-syn-related pathology, is the central to the pathogenetic processes observed in the brains of patients with PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). We are seeking an animal model with synucleinopathy that can comprehensively replicate these pathologies and adhere to suitable timeframes for preclinical research for positron emission tomography (PET) imaging studies.\u003c/p\u003e\u003ch2\u003eOBJECTIVE:\u003c/h2\u003e\u003cp\u003eTo evaluate the synucleinopathy mouse model that closely replicates key pathological features through immunohistochemistry (IHC) and to assess the application of this model for PET studies.\u003c/p\u003e\u003ch2\u003eMETHODS:\u003c/h2\u003e\u003cp\u003eAn adeno-associated virus (AAV) carrying the mutated human α-syn gene (AAV1/2-CMV-human-A53T α-syn) and preformed fibrils (PFF) of mutated recombinant human α-syn (S87N) were co-injected into the left substantia nigra (SN) of mouse brains. PET/CT imaging and IHC were performed at different time points post-injection to detect the key pathologies in the brain.\u003c/p\u003e\u003ch2\u003eRESULTS:\u003c/h2\u003e\u003cp\u003eThis model resulted in accelerated α-syn pathology, detectable as early as two weeks post-surgery, alongside DA neuron loss, microglial activation, reduced synaptic density, and impaired mitochondrial function within five weeks. The pathologies were localized, making the model suitable for PET imaging studies and/or PET ligand development. Both IHC and PET imaging confirmed the spatial relevance of these pathologies.\u003c/p\u003e\u003ch2\u003eCONCLUSIONS:\u003c/h2\u003e\u003cp\u003e This hybrid (AAV/PFF) mouse model provides an accelerated and localized platform for studying synucleinopathies such as PD, as well as for evaluating PET ligands for disease diagnosis and monitoring.\u003c/p\u003e","manuscriptTitle":"Accelerated and Localized Synucleinopathy in a Hybrid Mouse Model: Implications for Positron Emission Tomography Studies","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-30 12:06:14","doi":"10.21203/rs.3.rs-7821065/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T14:00:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-20T02:38:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-09T14:59:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"181441316679669484516359110316886337006","date":"2025-11-03T11:49:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337361603085899112397253345969393136746","date":"2025-10-19T03:53:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-16T14:02:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-16T09:59:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-13T16:49:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Imaging","date":"2025-10-09T22:05:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [npj Imaging](https://www.nature.com/npjimaging)","snPcode":"44303","submissionUrl":"https://submission.springernature.com/new-submission/44303/3","title":"npj Imaging","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8841888d-9ad5-4c97-957f-5fe17986c3e2","owner":[],"postedDate":"October 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57113284,"name":"Health sciences/Diseases"},{"id":57113285,"name":"Health sciences/Neurology"},{"id":57113286,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-01-12T15:54:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-30 12:06:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7821065","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7821065","identity":"rs-7821065","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}