Phosphorylation and Truncation of α-Synuclein do not trigger Parkinsonian Readouts in A53T-SNCA Mice

Phosphorylation and Truncation of α-Synuclein do not trigger Parkinsonian Readouts in A53T-SNCA Mice | 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 Phosphorylation and Truncation of α-Synuclein do not trigger Parkinsonian Readouts in A53T-SNCA Mice Annelore Anthonissen, Wilhelmus Drinkenburg, Patrik Verstreken, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9236452/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Parkinson’s disease is a multisystem disorder, in its prodrome characterized by a broad spectrum of non-motor symptoms, including olfactory deficits and REM sleep behavior disorder, that emerge years before the classical motor symptoms develop. Accordingly, a growing number of studies aim to generate mouse models exhibiting α-Synuclein pathology that recapitulate this prodromal phase and its progression to motor stages. This study investigated whether transgenic bacterial artificial chromosome mice carrying the human α-Synuclein gene - with the A53T point mutation, two single nucleotide polymorphisms and a Rep1 polymorphism - can capture features of prodromal and late motoric Parkinson’s disease through mutant α-Synuclein overexpression. Over a 24-month period, 20 heterozygous A53T-SNCA mice and 21 wild-type mice were longitudinally assessed for both non-motor and motor symptoms associated with Parkinson’s disease. EEG-EMG and local field potential recordings were performed to evaluate rapid eye movement sleep behavior disorder and stimulus-evoked neuronal activity disturbances, respectively. Additionally, we performed a behavioral phenotyping including the buried food seeking test and discriminations test for olfactory function, along with Rotarod and CatWalk assessments to evaluate motor performance. Terminal neuropathology was examined by immunohistochemistry, western blotting and a two-step direct immunoassay to correlate pathology with functional outcomes from the longitudinal study. Characterization of the final pathology in heterozygous A53T-SNCA mice revealed a SNCA transgene dose-dependent overexpression of α-Synuclein monomers, exhibiting Serine129 phosphorylation and C-terminal truncation, in the olfactory bulb, striatum and cortex. However, no SDS-resistant higher-molecular-weight α-Synuclein species [≥ 198kDa] were detected unlike those observed in the Parkinson’s disease brain sample. In addition, under our testing conditions, we were unable to identify early measurable signs of olfactory dysfunction or rapid eye movement sleep behavior disorder. Moreover, they maintained their motor performance up to 24 months, and showed no substantial loss of dopaminergic neurons, compared to wild-type mice. In summary, our results demonstrate that overexpression of Serine129 phosphorylated and C-terminally truncated α-Synuclein monomers in heterozygous A53T-SNCA mice is insufficient to drive mature fibrillar α-Synuclein aggregation, pronounced dopaminergic neurodegeneration or Parkinsonian (non-)motor symptoms. This transgenic model therefore highlights the limited ability of these post-translational modifications to initiate pathogenic processes relevant to prodromal and advanced Parkinson’s disease. Health sciences/Neurology Biological sciences/Neuroscience Parkinson’s disease α-Synuclein post-translational modifications transgenic mouse model olfactory dysfunction REM sleep behavior disorder Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Parkinson’s disease affects approximately 1% of the global population over 60 years of age, making it the second most common neurodegenerative disorder. Pathologically, it is characterized by the aggregation of misfolded intraneuronal α-Synuclein (α-Syn) deposits termed ‘Lewy Bodies’ and nigrostriatal dopaminergic neurodegeneration. These result in the progressive manifestation of motor impairments including bradykinesia, postural instability, resting tremor and rigidity. 1 – 3 Although clinical diagnosis focuses on motor impairments, growing evidence emphasizes the importance of non-motor symptoms that emerge years before motor dysfunctions appear in patients. 4 – 6 This pre-motor stage may offer a therapeutic window to test new disease-modifying approaches intended to modify or halt the progression of the disease early on. Additionally, the use of prodromal biomarkers may advance early identification of people at risk for developing Parkinson’s disease. This will attribute to a more accurate delineation of target populations for neuroprotective trials, facilitating the development of precision medicine-based therapies. 6 , 7 Recent evidence from multicenter studies indicates olfactory dysfunction and rapid eye movement sleep behavior disorder (RBD) as strong predictors of phenoconversion to parkinsonism, including Parkinson’s Disease, Dementia with Lewy Bodies and Multiple System Atrophy. The high prevalence of olfactory dysfunction and RBD in Parkinson’s disease, along with Braak’s hypothesis stating that the pathology initially targets the olfactory bulb and brainstem, supports their potential as predictive biomarkers. 1 , 8 – 10 Routinely used animal models do not replicate the progressive multisystem nature of the disorder, including its early non-motor phase and pathology beyond the nigrostriatal pathway. Focusing on models that reproduce the spatiotemporal prodromal-to-late progression of Parkinson’s disease may enhance insights into the pathogenesis and strengthen translational validity. 11 , 12 Therefore, we evaluated a prodromal Parkinson’s disease mouse model described by Taguchi et al. 13 that harbors a bacterial artificial chromosome (BAC) with the human SNCA gene in which the A53T mutation, two risk-associated single-nucleotide polymorphisms (rs11931074, rs3857059) and a Rep1 dinucleotide repeat polymorphism were introduced. According to their study, this model exhibits α-Syn pathology across multiple brain regions, dopaminergic neurodegeneration, early signs of olfactory dysfunction and RBD, but no motor impairments at the age of 18 months. 13 In our study, we investigated whether SNCA transgene dose-dependent overexpression of α-Syn, including Serine129 phosphorylated and C-terminally truncated forms, was sufficient to cause key pathological and functional readouts featuring prodromal-to-late Parkinson’s disease in heterozygous A53T-SNCA BAC mice ( SNCA A53T/− BAC) over a 24-month period. Results Copy number variation in heterozygous A53T-SNCA BAC Mice To ensure genetic homogeneity, we quantified human SNCA copies in our longitudinal cohort of 20 SNCA A53T/- BAC mice using ddPCR ( Fig. 1 ) . SNCA copies ranged from 17.8 to 58.5 (median 32.76), and mice were therefore divided into low ( 32.76 SNCA copies) SNCA A53T/- groups. The average copy number of human SNCA in low SNCA A53T/- group (21.84 ± 1.17) was significantly lower (difference: 31.20 ± 1.90, P < 0.0001) compared to high SNCA A53T/- group (53.04 ± 1.39). The stratification of the transgenic group allowed us to assess whether variability in SNCA transgene dose impacted α-Syn expression and the onset or severity of functional readouts related to Parkinson’s disase. Overexpression of pS129 and truncated α-Syn do not induce fibrillar aggregation in SNCA A53T/- mice To assess α-Syn pathology in 24-month-old SNCA A53T/− BAC mice, we performed SDS-PAGE Western blotting on pooled brain homogenates using pS129-α-Syn (EP1536Y) and α-Syn (MJFR14 & Syn-1) antibodies. pS129-α-Syn monomers [~ 15kDa] increased in SNCA transgene dose-dependent manner in olfactory bulb, striatum and cortex, with the highest levels in the olfactory bulb, while no increase was detected in brainstem (Fig. 2 a). In contrast to the human Parkinson’s disease brain sample, no comparable higher-molecular-weight (HMW) pS129-α-Syn species [≥ 198kDa] were observed ( Fig. 2 a ) . This was confirmed by the MJFR13-81A immunoassay, in which interpolated values for pooled olfactory bulb, striatum and cortex homogenates from SNCA A53T/− BAC mice fell at or below the limit of quantification ( Fig. 2 b ) . Additionally, proteinase K treatment eliminated monomeric pS129-α-Syn in all regions of SNCA A53T/− BAC mice, whereas both monomeric and HMW α-Syn persisted in the human Parkinson’s disease brain sample (Fig. 2 c), indicating that pathological α-Syn species associated with Parkinson’s disease are proteinase K-resistant. To assess the presence of pathological α-Syn aggregates in SNCA A53T/− BAC mice, MJFR14 solely revealed a SNCA transgene dose-dependent increase in monomeric α-Syn levels [~ 15kDa] across all regions. Given the denaturing conditions of SDS-PAGE, monomeric α-Syn signals could theoretically arise from dissociation of fragile, non-covalent early oligomeric forms. Nevertheless, the SDS-resistant HMW α-Syn aggregates [≥ 198kDa] observed in human Parkinson’s disease brain sample were not detected in SNCA A53T/− BAC mice ( Fig. 2 d ) . Furthermore, Syn-1 showed a SNCA transgene dose-dependent rise in monomeric α-Syn [~ 15kDa] across all brain regions and additionally revealed C-terminally truncated α-Syn species [< 15kDa] in SNCA A53T/− BAC mice, most abundant in olfactory bulb, striatum and cortex (Fig. 2 e). Overall, SNCA A53T/− BAC mice displayed region- and SNCA transgene dose-dependent elevations in pS129 and C-terminally truncated α-Syn monomers, but no evidence of SDS-resistant HMW α-Syn species [≥ 198kDa] comparable to those detected in the human Parkinson’s disease brain sample at 24 months. No signs of olfactory dysfunction in SNCA A53T/- mice Olfactory dysfunction may signal underlying α-Syn pathology many years before a clinical diagnosis of Parkinson’s disease. 20 – 23 Therefore, we evaluated whether SNCA A53T/− BAC mice exhibit impairments in olfactory detection and discrimination. As illustrated in Fig. 3 a, the buried food seeking test performed at 10 and 24 months showed no disability in olfactory detection in SNCA A53T/− BAC mice compared to controls. Additionally, olfactory discrimination – measured as the difference in sniffing time between water and vanilla - was not significantly altered in SNCA A53T/− BAC mice compared with controls at 14 months ( Fig. 3 b ) . Moreover, LFP of the olfactory bulb recorded during the discrimination paradigm showed no significant deviations in power spectral density between genotypes ( Fig. 3 c ) across physiologically relevant frequency bands: theta [4-8Hz] ( Fig. 3 d ) , alpha [8-12Hz] ( Fig. 3 e ) , beta [13-30Hz] ( Fig. 3 f ) and gamma [30-100Hz] ( Fig. 3 g ) . Given that olfactory performance can be affected by various confounding factors such as anxiety or depression, we performed the light-dark test. Cumulative duration spent in both compartments (Fig. 3 h ) , time in light compartment ( Fig. 3 i ) and number of entries to light compartment ( Fig. 3 j ) revealed no differences between SNCA A53T/− BAC mice and controls. Hence, olfactory performance was likely not affected by differences in anxiety levels. Furthermore, we assessed pS129-α-Syn levels and found significant SNCA transgene dose-dependent labeling in the olfactory bulbs of 18-month-old SNCA A53T/− BAC mice compared to controls ( Fig. 3 k ) . This immunoreactivity appeared to persist over time, as a separate cohort of 4-month-old wild-type and SNCA A53T/− BAC mice showed similar pattern and intensity of the immunoreactivity (Supplementary Fig. 5) . In wild-type mice, low physiological levels of pS129-α-Syn are found in the anterior olfactory bulb, mitral cell layer, external plexiform layer and glomerular layer. In contrast, SNCA A53T/− BAC mice exhibit higher intensity of pS129-α-Syn in these regions, along with immunoreactivity in the inner plexiform layer, granule cell layer and subependymal zone. Interestingly, upon incubation with proteinase K, no pS129-α-Syn was detected in wild-type mice, whereas SNCA transgene dose-dependent immunoreactivity in SNCA A53T/− BAC mice persisted in regions that normally contain physiological pS129-α-Syn, but disappeared from the inner plexiform layer, granule cell layer and subependymal zone. However, the interpretation that this reflects the presence of PK-resistant pS129-α-Syn species should be made cautiously, as suboptimal proteolysis at the employed PK dose could result in residual immunoreactivity. This caution is supported by western blot data ( Fig. 2 c ) , showing that pS129-α-Syn in SNCA A53T/− BAC homogenates was fully degraded under PK conditions. No early REM sleep behavior disorder-like phenotype observed in SNCA A53T/- mice Considering the emerging link between prodromal RBD and the phenoconversion to Parkinson’s disease 24 – 27 , we performed a longitudinal assessment of motor activity during REM sleep in SNCA A53T/− BAC mice. At various time points, we assessed their 24-hour sleep-wake architecture using EEG-EMG recordings, complemented by passive infrared monitoring and synchronized video tracking. SNCA A53T/− BAC mice showed no significant differences in total wake, NREM or REM sleep duration compared with controls (Fig. 4 a–c). This indicates that subsequent analyses of RBD-like behavior are unlikely to be influenced by REM duration. Since RBD is characterized by REM sleep without atonia (RSWA), we examined whether SNCA A53T/− BAC mice exhibited increased muscle activity during REM sleep. We used two calculation approaches: (1) an automated detection of REM epochs with EMG REM power exceeding three standard deviations of EMG NREM power ( Fig. 4 d ) and (2) a manual screening of REM epochs with elevated EMG amplitude and visible movement ( Fig. 4 e ) . Neither approach showed a significant increase in muscle activity in SNCA A53T/− BAC mice, irrespective of their transgene dose, compared to wild-type mice at any time point. Additionally, EEG-EMG traces showed comparable REM-associated movements across wild-type, low SNCA A53T/− and high SNCA A53T/− BAC mice (Fig. 4 f). This suggests that the movements in this study may not be considered abnormal events featuring an RBD-like phenotype but rather represent normal biological phenomena during REM sleep. Collectively, the lack of increased EMG activity indicates that SNCA A53T/− BAC mice do not exhibit RBD-like behavior. Furthermore, the sublaterodorsal tegmental nucleus, a region in the brainstem that regulates muscle tone during sleep-wake cycles, 28–30 showed no deposition of pS129-α-Syn in SNCA A53T/− BAC mice, potentially corroborating with the absence of RBD-like features ( Fig. 4 g ) . Preserved motor performance in 24-month-old SNCA A53T/- mice The main pathological feature of Parkinson’s disease involves the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. These neurons project through the nigrostriatal pathway to the striatum resulting in a striatal depletion of dopamine that dysregulates motor activity. 31 , 32 Consequently, bradykinesia, rigidity, postural instability and resting tremor are considered cardinal signs of Parkinson’s disease. 3 , 33 To assess progressive motor dysfunctions in SNCA A53T/− BAC mice, we conducted the Rotarod motor performance test and Catwalk XT gait analysis at multiple timepoints up to 24 months. Temporal and spatial gait parameters showed no significant deviations between SNCA A53T/− BAC and wild-type mice at 24 months (Fig. 5 a-c). Additionally, latency to fall in the accelerating Rotarod test did not differ between SNCA A53T/− BAC mice and wild-type mice (Fig. (5d) . Since no motoric readouts were detectable yet, we evaluated whether neurophysiological alterations might occur in these mice, potentially preceding a future overt motor dysfunction. Therefore, we analyzed LFPs recorded in primary motor cortex ( Fig. 5 e ) and substantia nigra ( Fig. 5 j ) during active wake stages over a 24-hour period. At 18 months, LFPs in SNCA A53T/− BAC mice revealed no significant changes in mean power across different physiological frequency bands, including theta [4-8Hz], alpha [8-12Hz], beta [13-30Hz] and gamma [30-100Hz], compared to wild-type mice ( Fig. 5 f-i & Fig. 5 k-n ) . Immunohistochemical analysis of striatum in a distinctive cohort of 18-month-old SNCA A53T/− BAC mice revealed SNCA transgene dose - dependent extracellular and neuritic pS129-α-Syn depositions compared to wild-type mice ( Fig. 5 o ) . To assess dopaminergic neurodegeneration, we evaluated the survival of tyrosine hydroxylase-positive neurons in the striatum, nigrostriatal pathway and substantia nigra of SNCA A53T/− BAC mice after 24 months. Similar intensity averages were observed across all groups, as well as an equal ratio of immunoreactive area over the regions of interest (Fig. 6 ). This indicates that SNCA A53T/− BAC mice in this study do not exhibit robust dopaminergic neurodegeneration after two years, despite the presence of monomeric pS129 and C-terminally truncated α-Syn in the striatum and midbrain as observed through immunohistochemistry ( Fig. 5 o ) and western blotting ( Fig. 2 a & Fig. 2 e ) . Discussion In this study, we evaluated the translational validity of heterozygous A53T-SNCA BAC mice as a model capturing prodromal-to-late Parkinson’s disease progression. This line carries the human SNCA gene with the A53T point mutation, two risk-associated single nucleotide polymorphisms and the Rep1 repeat. 13 , 34 Although previous reports described Parkinsonian phenotypes in this model, 13,34,35 we did not observe such features under our experimental conditions. Despite copy number variation of the mutant SNCA gene across the cohort, even high-copy mice remained free of non-motor and motor deficits and showed no electrophysiological abnormalities over a 24-month period. At the molecular level, SNCA transgene dose-dependent increases in pS129 and C-terminally truncated α-Syn species was observed in olfactory bulb, striatum and cortex; however, these changes were not accompanied by SDS-resistant HMW α-Syn species [≥ 198kDa], similar to those observed in Parkinson’s disease brain sample. Taken together, our findings suggest that the A53T mutation together with additional risk-associated polymorphisms in human SNCA gene are insufficient to drive the pathogenic processes underlying pre-motor or motor manifestations in SNCA A53T/− BAC mice within a 2-year window. The lack of mature fibrillar aggregates and Parkinsonian readouts further indicates that Ser129 phosphorylation and C-terminal truncation of α-Syn alone do not promote overt α-Syn aggregation in vivo , or, alternatively, one could argue that the observed α-Syn overexpression does not surpass the threshold required for initiating pathogenic cascades. Copy number variation in heterozygous A53T-SNCA BAC mice We observed a variability in copies of the mutant SNCA gene in our heterozygous A53T-SNCA BAC mice. 36 This was surprising since all mice originated from a breeding pair with a heterozygous A53T-SNCA BAC parent preselected for high mutant SNCA dose. This underscores the importance of monitoring transgene copy number variation in BAC-based animal models. 37 , 38 The copy number variation across our cohort is unlikely to result from random integration of the BAC construct, as a consistent 1:1 segregation ratio was maintained during breeding. Instead, this variability may reflect recombination of the large BAC construct during meiosis, resulting in diverse copy numbers or by the instability of the Rep1 dinucleotide repeat polymorphism that plays a pivotal role in the transcriptional regulation of the human SNCA gene. 39 Future studies should focus on advancing BAC constructs to enable more targeted transgene delivery and to mitigate genetic drifts while maintaining sufficient SNCA expression to induce pathological changes. pS129 and truncated α-Syn overexpression fails to induce fibrillar aggregation in SNCA A53T/- mice Our study showed that the increase of mutant SNCA copies was positively correlated with the overexpression of monomeric α-Syn and the abundance of Serine 129 phosphorylation and C-terminal truncation across various brain regions in 2-year-old SNCA A53T/− BAC mice. It has been demonstrated that these PTMs alter the propensity of α-Syn to promote aggregation by affecting structure and conformation, and consequently its solubility. 40 – 43 However, within the context of our experiments, we did not detect HMW (pS129-) α-Syn species [≥ 198kDa] that would imply the presence of mature fibrillar (pS129-) α-Syn aggregates resembling those of the Parkinson’s disease brain sample. Possibly, mice with highest mutant SNCA expression in this study may still have insufficient levels to induce α-Syn aggregation. 35 , 44 Another explanation, of a technical nature, could be the use of SDS-PAGE western blotting to detect HMW α-Syn species. Since SDS disrupts non-covalent interactions within aggregates, it could cause dissociation into smaller and more fragile fragments, masking the presence of early oligomeric species. In line with this, Taguchi et al. reported the presence of oligomeric α-Syn using O1 immunostaining, but did not detect fibrillar aggregates with the fibril-specific F2 antibody 13 , supporting the notion that α-Syn species in this model do not resemble the SDS-resistant fibrillar aggregates featuring Parkinson’s disease. To evaluate the potency of the present forms of pS129-α-Syn to promote aggregation in this model, we assessed their resistance to proteinase K. While pathogenic pS129-α-Syn species from the Parkinson’s disease brain sample remained resistant to proteinase K digestion due to their insolubility, all pS129-α-Syn were eliminated in the model. This suggests that pS129-α-Syn species in our mice are vulnerable for digestion due to their soluble nature, explaining their inability to induce fibrillar aggregation. 44 – 47 Interestingly, our observations are in line with previous studies highlighting the physiological role of PTMs - Serine 129 phosphorylation and C-terminal truncation - and the lack of consensus on their pathogenic contribution to aggregation. 48 – 53 A recent study showed that after injection of α-Syn preformed fibrils into the striatum of mice, pS129-α-Syn became apparent four weeks post-injection, suggesting that phosphorylation occurs after the initial seeding and protein aggregation. 54 Additionally, SNCA A53T/− BAC mice also demonstrated C-terminal truncation of α-Syn, which is believed to promote aggregation as it removes protective regions. 43 , 54 However, the mice did not exhibit HMW α-Syn species, challenging its causative role in aggregation. Recent work by Mahul-Mellier et al. 55 has shown that C-terminal truncation primarily acts as a post-fibrillization regulator that drives fibril remodeling and maturation into Lewy body–like inclusions, rather than initiating aggregation. This suggests that Ser129 phosphorylation and C-terminal truncation could be epiphenomena that accompany aggregation, rather than playing direct causal roles in pathogenesis. Lack of phenotypic readouts related to Parkinson’s disease in SNCA A53T/- mice Behavioral assessments with corresponding neurophysiological recordings revealed no early signs of REM sleep behavior disorder, olfactory dysfunction or late motor impairments in our SNCA A53T/ − BAC cohort. Notably, this absence of prodromal non-motor symptoms contrasts with the findings reported by Taguchi et al. 13 Nevertheless, our functional readouts may be attributable to the absence of α-Syn aggregates in the relevant regions. Similarly, previous work by Moceri et al. 35 showed no early olfactory defect in A53T-SNCA BAC mice, despite immunohistochemical analyses revealing high levels of pS129-α-Syn in the olfactory bulb. These findings strengthen the yet-unknown biological role of pS129-α-Syn in the olfactory bulb, where it is abundantly present in the healthy brains of both humans and mice. 48 , 56 A clinicopathological study by Tremblay et al. 57 revealed that pS129-α-Syn deposition in the olfactory bulb of humans with age-related olfactory decline was not found to correlate with their olfactory performance, suggesting pS129-α-Syn does not seem to independently drive olfactory deficits. Nevertheless, a potential shortcoming of this study involves the use of conventional olfactory paradigms to evaluate olfactory performance. They provide a robust interpretation of whether mice can discriminate or perceive odors well but lack sensitivity to more subtle deficits. Therefore, future studies may benefit from using an automated olfactometer that standardizes olfactory stimulation by controlling odor concentrations, release time and flow rate. 58 , 59 Furthermore, the absence of increased muscle activity during REM sleep in SNCA A53T/− BAC mice may reflect a lack of pS129-α-Syn deposition in the sublaterodorsal tegmental nucleus, a region implicated in the pathogenesis of RBD in prodromal Parkinson’s disease. 60 – 63 Interestingly, Shen et al. 28 demonstrated that inoculating α-Syn fibrils into the SLD of mice triggers RBD-like behavior and later Parkinson’s disease, supporting a pathogenic role for \(\:\alpha\:\) -Syn pathology in RBD. Nevertheless, characterizing RBD-like behavior in mice requires standardization, as the current diversity of assessment methods hinder reproducibility and reliable interpretation. Existing studies often infer RBD-like phenotypes from automated EEG-EMG quantification. 28 , 60 , 64 Although these approaches provide an indication of muscle tone, they should be supplemented by synchronized videos to distinguish pathological phenomena from physiological movements or potential artefacts during REM sleep. In this study, the EEG-EMG traces with synchronized video show that the movements during REM sleep in SNCA A53T/− BAC mice are identical to those in wild-type controls, indicating that these events are physiological rather than genotype-specific RSWA-like events. Standardized protocols combining EEG–EMG, synchronized video and non-transgenic controls are therefore essential to avoid misclassification and to determine whether mice can truly exhibit RBD-like behavior. To be translationally relevant for Parkinson’s disease, a mouse model must exhibit nigrostriatal dopaminergic neurodegeneration and motor impairments. In this study, SNCA A53T/− BAC mice did not exhibit a reduction in TH-immunoreactivity – marker for dopaminergic neurons - along the nigrostriatal system at 24 months. Notably, the subtle reduction in TH-positive neurons reported by Taguchi et al. 13 at 18 months was identified using a more spatially resolved analysis across the entire midbrain, which may explain the discrepancy with our present findings. Additionally, these mice showed no evidence of altered neuronal activity in the substantia nigra or impaired motor performance in the CatWalk and RotaRod assessments. These findings corroborate those of Taguchi et al. 13 and confirm that aging beyond 18 months does not lead to progressive motor impairments as previously hypothesized. Previous studies using transgenic mice overexpressing human α-Syn have similarly reported neither locomotor deficits nor clear dopaminergic neurodegeneration, despite substantial accumulation of (pS129-) α-Syn across multiple brain regions. 65 – 67 Together, these findings highlight the need to identify additional mechanisms that drive late Parkinsonian motor dysfunction in mouse models. In this 24-month study of heterozygous A53T-SNCA BAC mice, we assessed α-Syn pathology, neural activity and behaviors relevant to both prodromal and advanced Parkinson’s disease. We observed SNCA transgene dose-dependent overexpression of Serine129 phosphorylated and C-terminally truncated α-Syn across the olfactory bulb, striatum and cortex. Despite these molecular alterations, we did not detect mature fibrillar α-Syn aggregation, prodromal features including olfactory impairments or RBD-like manifestations, nor a progression to pronounced dopaminergic neurodegeneration or motor deficits. Collectively, these findings indicate that overexpression of pS129 and C-terminally truncated α-Syn alone is insufficient to drive aggregation or functional readouts associated with Parkinson’s disease within 24 months. This highlights the need to identify additional factors required to recapitulate prodromal-to-late motoric Parkinson’s disease in mouse models. Methods Animal Husbandry Animal housing, handling and experiments were strictly in accordance with the international directives of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by the Ethical Committee for Animal Experimentation at Johnson & Johnson. ARRIVE guidelines were used for reporting in vivo research. 14 Animals were housed on a 12h dark/light cycle in Emerald EM500 cages with enrichment, food (SAFE® A05) and water access ad libitum , except for fasting experiments. Behavioral cohorts were single housed; molecular cohorts were group housed. Environmental conditions such as temperature, humidity and pressure were automatically controlled to maintain respective levels of 22 ± 2°C, 55 ± 10% and 4 Pa. All behavioral experiments were conducted in the dark to respect mice’s nocturnal activity. Animal Breeding & Genotyping Cryopreserved SNCA A53T/- BAC embryos (N10) with high SNCA transgene dose were kindly provided by Prof. Dr. Hodaka Yamakado (Kyoto University). 13 SCNA A53T/- BAC males and wild-type C57BL/6J females were paired at the Transgenic Rodent Facility of Johnson & Johnson, and N15 offspring were used for experiments. Genotyping was performed on tail biopsies using the Bio-Rad QX200 Droplet Digital Polymerase Chain Reaction (ddPCR) system (IGBMC) to assess A53T-SNCA copy number variation. Genomic DNA was extracted with the DNeasy Blood & Tissue Kit (Qiagen) and quantified via NanodropTM spectrophotometer (ThermoFisher Scientific). ddPCR reactions contained 10ng gDNA, 2X ddPCR supermix, 20X TaqMan probes for human SNCA and mouse Ap3b1 and nuclease-free water (InvitrogenTM). Droplets were generated, read on the QX200 system and analyzed with QuantaSoft Analysis Pro. SNCA copy number was calculated as the SNCA / Ap3b1 ratio and multiplied for diploidy. Animal Cohorts Longitudinal assessments - including behavioral testing, EEG-EMG and local field potential (LFP) recordings - were performed on wild-type C57BL/6J mice ( n = 21) and SNCA A53T/− BAC mice ( n = 20), which were divided into low-copy (low SNCA A53T/− : n = 13) and high-copy (high SNCA A53T/− : n = 7) groups. At 24 months, mice were sacrificed, with the left hemisphere used for immunohistochemistry and the right hemisphere for western blotting and 2-step direct immunoassay. Additional cohorts were aged to 4 months (wt: n = 8; SNCA A53T/− : n = 8) and 18 months (wt: n = 8; SNCA A53T/− : n = 8) for age-dependent pathology mapping by immunohistochemistry. Male and female outcomes were analyzed separately but reported together, as no significant gender differences were observed. Supplementary Fig. 1 illustrates the experimental design of this study. Stereotaxic Electrode Implantation Surgeries were performed on 2-month-old mice after induction with 5% isoflurane anesthesia, with morphine (2mg/kg) and Metacam® (5mg/kg) for analgesia. Isobetadine (Meda Pharma SA) and 70% Ethanol was applied to disinfect the surgical site, and eye ointment (Opticorn A, EcuPhar BV) was used to prevent dehydration during surgery. Once mice were fixed in the stereotaxic frame, anesthesia was maintained by 2%-2.5% isoflurane and body temperature was regulated by Harvard homeothermic monitoring system. Two stainless-steel EEG electrodes (7N51465T5TLT, 51/46 Teflon Bilaney) were implanted over the left frontal cortex (AP + 2.3mm, ML -1.5mm) and parietal cortex (AP -1.5mm, ML -1mm). A reference electrode was placed 1mm posterior to lambda, and a ground electrode was positioned centrally to reduce noise. Four polyamide-coated stainless-steel depth electrodes (100µm diameter with a blunt-tip, customized by Peira bvba, Komax) were implanted in the right hemisphere at following coordinates (from dura): olfactory bulb (AP + 4.5mm, +ML 1.2 mm, DV -0.8mm), primary motor cortex (AP + 1.5mm, ML + 1.6mm, DV -0.7mm), CA1 (AP -1.94mm, ML + 1.2mm, DV -1.15mm) and substantia nigra (AP -3.28mm, ML + 1.2mm, DV -4.3mm). Fine-wired electrodes were inserted in two trapezius muscles for EMG. The electrode configuration is shown in Supplementary Fig. 2 . All electrodes were secured with dental cement (Relyx Unicem 2 cement, 3M) and connected to a 10-channel headmount. Skin was closed with tissue-adhesive glue (Vetbond, 3M). Mice received 0.9% NaCl post-recovery, were closely monitored for 1 hour, and then housed in a heated cabinet with DietGel® 76A for 24 hours before daily checks for four days. Video-assisted Electrophysiological Recording After 2 weeks of recovery, mice were placed in sound-attenuated chambers with enrichment, food and water access ad libitum , and maintained under similar housing conditions. Each mouse underwent 48 hours of continuous recording, including a 24-hour habituation period, under a maintained light-dark cycle. Two video cameras (uEye CP, IDS Imaging GmbH) captured top and lateral views of behavior. EEG, LFP and EMG signals were acquired at 512Hz using the BioSemi system and stored in BDF format. General motor activity was also monitored via two passive infrared detectors placed above each cage. Electrophysiological Data Analysis Raw Data Processing BDF files were imported into a LabVIEW-based analysis tool (National Instruments). Data were pre-processed with a 50Hz-Notch filter and a 1-200Hz band-pass Butterworth filter. Recordings were referenced to the reference electrode and grounded to reduce unwanted noise and interference from the environment. Visual inspection facilitated quality check and potential exclusion of signals showing flat lines indicating disconnection, high levels of artifacts, or widespread interference across electrodes. Vigilance State Analysis Sleep-wake states were scored in 2s-epochs over 24 hours of EEG-EMG-video recordings using a semi-automated LabVIEW-based tool (National Instruments). A machine learning algorithm provided initial scoring, followed by manual verification of the EEG-EMG traces and synchronized videos to accurately assign active wake, quiet wake, NREM sleep and REM sleep. Active wake was characterized by low-amplitude EEG with beta activity [13-30Hz], high EMG and PIR > 0.25; quite wake by low-amplitude EEG with alpha activity [8-12Hz], low EMG and PIR < 0.25. NREM sleep showed high-amplitude EEG with delta activity [1Hz-5Hz], low EMG and PIR < 0.25, whereas REM sleep displayed high-amplitude EEG with theta activity [4-8Hz], flat EMG and PIR < 0.25. Representative traces are shown in Supplementary Fig. 3 . Time spent in each vigilance stage [min] was quantified per hour. EMG Signal Quantification EMG recordings from trapezius muscles were used to assess REM sleep without atonia (RSWA), a primary feature of RBD-like behavior. Therefore, REM sleep muscle activity was evaluated using two approaches: (1) manual scoring of REM epochs featuring elevated EMG amplitude and visible movement, and (2) automated analysis of EMG power per REM epoch using Fast Fourier Transform, normalized to EMG NREM power. REM epochs with EMG power exceeding three standard deviations above EMG NREM power were classified as showing increased REM sleep muscle activity. The manual analysis acts as a control to prevent overestimation or misinterpretation of REM sleep muscle activity caused by EMG artifacts, while the automated analysis minimizes experimenter bias inherent to manual scoring. Power Density Analysis Power density spectra were derived from 24-hour LFP recordings using Fast Fourier Transformation with a Hanning window function by transforming the time-domain signals into a frequency-domain spectrum. This was performed through a customized analysis LabVIEW-tool (National Instruments). The power [dB] was calculated as the square of EEG magnitude [µV] on a logarithmic scale. With a frequency resolution of 0.5Hz, the power was plotted across the frequency range from 0.5Hz up to 100Hz. Behavioral Assessments Olfactory Buried Food Test Following an overnight fasting, the buried food-seeking test investigated the ability of mice to detect the vanilla odor originating from a cookie buried beneath 3cm clean bedding. 15 – 17 Latency time [s] to retrieve the buried cookie was recorded from videos using a stopwatch. A habituation period of 2 hours allowed the mice to accommodate to a clean cage with 3cm clean bedding. Olfactory Discrimination Test Olfactory discrimination was assessed by simultaneously presenting two filter papers containing water (neutral) and vanilla aroma (Vahiné, 1:100; attractive) for 2 minutes in the home cage placed inside sound-attenuated chambers. 16 , 17 Cumulative sniffing duration [s] directed to each stimulus was measured from video recordings using a stopwatch. Discrimination ability was defined by the difference in sniffing time [s] between water and vanilla. During testing, mice were wired to enable concurrent LFP recordings. Prior to the assay, mice were habituated to experimental conditions for 2 hours. Light-Dark Test The light-dark transition test, which exploits the innate aversion of mice to illuminated areas, was used to assess exploratory behavior and anxiety. 18 , 19 The apparatus (40 x 40 cm) consisted of an equally sized light compartment (1000 Lux) and dark compartment connected by a small opening and equipped with infrared beam sensors for movement tracking. Mice were placed in the dark chamber and allowed to explore freely for 15 minutes. EthoVision XT Software (version 17, Noldus) automatically quantified time spent in each compartment [s] and the number of transitions. Accelerating RotaRod Motor Performance Test To longitudinally investigate impairments in motor coordination and postural instability, mice were placed on an accelerating RotaRod apparatus (CT-ENV-575M-X4, Med Associates Inc. St. Albans). All animals underwent a training trial of five minutes before the testing protocol. The testing protocol involved three subsequent trials in which animals ran for five minutes straight on a from 4-to-40rpm accelerating rod with 15 minutes time interval. The latency [s] for each mouse to fall from the apparatus was recorded and averaged across three trials as indicator for motor coordination. CatWalk XT Gait Analysis Gait abnormalities were assessed using the Catwalk XT system (Noldus). Mice freely transverse an enclosed gateway on an illuminated glass plate that captures pawprints with a high-speed video camera positioned under the gateway. Habituation to the CatWalk XT system involved allowing mice to freely transverse the gateway. The following day, mice were trained by completing three compliant runs, each with a maximum speed variation of 60%. On the experimental day, a minimum of five consecutive runs with a maximum speed variation of 25% were acquired to ensure reliable data interpretation. Catwalk XT 10.6 software enabled the calculation of an extensive range of spatial and temporal measurements. Immunohistochemistry Mouse brains were collected through anaesthetized decapitation and fixed in 10% buffered formalin for 24 hours. Samples underwent automated processing steps: 30min in formalin, 30min in water, 1h in 70% ethanol, 1h30 in 80% ethanol, 30min in 96% ethanol, 1h30 in 96% ethanol, 1h30 + 2h in 100% ethanol and 1h + 2h in xylene. Afterwards, samples were embedded in paraffin wax at 63°C and cooled. Sections of 5µm were sliced using a Leica RM2255 microtome and dried at 50°C for 24 hours. Upon staining, sections were deparaffinized by sequential immersion steps: 2 x 10min xylene, 5min in 100% ethanol, 5min in 90% ethanol, 5min in 70% ethanol, 5min in 50% ethanol and 5min in water. Antigen retrieval was performed with 88% formic acid in distilled water for 20min, followed by 2.5µg/mL proteinase K (Roche, 03115836001) in PBS for 30min at room temperature. Endogenous peroxidase was blocked with 3% hydrogen peroxidase (DAKO, S2023) for 5min. Next, sections were incubated with primary antibodies, washed in wash buffer (DAKO, S3006) and followed by 30min incubation with secondary antibodies ( Supplementary Table 1 ). Target antigens were visualized during 10min incubation of DAB + chromogen (DAKO, K3468) followed by counterstaining with hematoxylin. Dehydration was conducted by immersion of 2min in 90% ethanol, 2 x 2min in 100% ethanol and 2 x 2min in xylene. Finally, slides were scanned by NanoZoomer® S60vs2 digital slide scanner (Hamamatsu, C16600-01) using brightfield microscopy with 40x resolution. Image Analysis Images were acquired through HALO Link and analyzed with VisioPharm® Version 2024.07 x64, an AI-driven image analysis software. Automated image classification was performed using Analysis Protocol Package (APP) created from manually annotated features (eg. PK-resistant p-α-Syn neural cells, TH-positive cells) on a subset of representative samples. The APPs were trained with a U-Net deep learning model for pixel-wise image segmentation, enabling precise feature identification. After manual validation of model sensitivity and specificity, APPs were applied to all images to detect specific features. Output variables from VisioPharm® were further analyzed in GraphPad Prism 10. Biochemical Assays Tissue Homogenization and Bicinchoninic Acid (BCA) Analysis Right olfactory bulb, striatum, brainstem and cortex were micro-dissected from 24-month-old mice and homogenized in RIPA buffer (comprising 50 mM Tris HCl, 150 mM NaCl, 1 mM MgCl2, 1.0% NP-40 (v/v), 0.5% sodium deoxycholate (w/v), 1 mM EDTA, 0.1% SDS (w/v), pH 7.4, Sigma, R0278) supplemented with protease inhibitor (Roche, 4693159001) and phosphatase inhibitors (Roche, 4906837001). After centrifugation at 20.000g for 5min at 4°C, supernatant was collected and protein concentration was determined using a BCA kit (BCA Protein Assay-Kit, Thermofisher). Samples were stored at -80°C prior to further analysis. SDS-PAGE Western Blotting Homogenized samples were incubated for 30min at 37°C with 1:50 proteinase K (Roche, 03115836001). For SDS-PAGE western blotting, 20µg of the PK-(un)treated samples were dissolved in NuPAGE LDS sample buffer (4X, Invitrogen™) supplemented with NuPAGE Sample Reducing Agent (10X, Invitrogen™) and boiled at 75°C for 5min. After separating on a NuPAGE 4–12% (w/v) gradient gel (Invitrogen™), proteins were transferred to a nitrocellulose membrane (BioRad) using the TransBlot Turbo system. For detection of α-Syn, the membranes were treated with 4% (w/v) PFA in PBS for 30 min at room temperature. After washing in TBS-T, the membranes were blocked for 1h using 5% skim milk (w/v) in TBS-T. They were then incubated overnight at 4°C with primary antibodies ( Supplementary Table 1 ). After washing, the membranes were exposed to horseradish peroxidase-conjugated secondary antibodies for 1h at room temperature ( Supplementary Table 1) . Super Signal West Dura (Thermo Fisher Scientific) was applied and chemiluminescence was imaged by Amersham™ 600 Imager (GE Healthcare). Target protein expression was quantified by normalizing band intensities to β-actin using ImageQuantTL software. Meso Scale Discovery Two-Step-Direct Immunoassay Total pS129-α-Syn aggregates were quantified using MJFR13-81A immunoassay on a 96-well SECTOR® plate (MSD, L15X1-3). Plates were coated overnight at 4°C with 0.5µg/mL MJFR13 antibody (Abcam, 168381) and washed afterwards with phosphate buffered saline (Gibco DPBS, Thermo Fisher Scientific, MA, USA) and 0.05% (v/v) Tween-20 (VWR, PA, USA). Blocking was performed with 0.1% Casein buffer (Thermo, 37528) for 2 hours at room temperature. Calibrator (recombinant pS129-α-Syn PFF) and pooled samples were thawed, diluted in RIPA buffer (Sigma, R0278) supplemented with complete protease (Roche, 4693159001) and PhosStop phosphatase inhibitors (Roche, 4906837001) and added in duplicates for overnight incubation at 4°C. After washing, 1µg/mL 81A antibody (Biolegend, 825701) was incubated for 2 hours at room temperature, followed by 1µg/mL of secondary Sulfo-TAG labeled secondary antibody (Meso Scale Discovery, R32AB-1) for 1 hour. After washing, 1X MSD Read Buffer T was added, and signals were measured with the MSD S600 Sector Imager. Concentrations were calculated using a 4-parameter logistic curve fit with 1/Y 2 weighing, excluding calibration points with relative error exceeding 20%. Final curve fit was used to interpolate raw data and determine sample concentrations [ng/mL]. Data points falling outside the linear range of the curve fit, or at or below lower limit of quantification - defined as mean blank signal plus 10 standard deviations of blank signal – were excluded from analysis. Statistical Analysis Statistical analyses were conducted in GraphPad Prism version 10. To verify if Markov assumptions were met, Shapiro-Wilk test and QQ-plot were performed to check normality of the residuals. Levene’s test and pattern recognition in residuals versus predicted values plots were used to check for homo- or heteroscedasticity. Two-tailed T-tests or ANOVA tests were used when assumptions were met; otherwise, non-parametric tests were applied. Additionally, post-hoc tests were applied to explore significant differences between groups. A P -value < 0.05 was considered to indicate a statistically significant difference. Data Availability The authors confirm that the data supporting the findings of this study are available within the article and its supplementary material. Abbreviations α-Syn (alpha-Synuclein), BAC (bacterial artificial chromosome), RBD (Rapid Eye Movement Sleep Behavior Disorder), PTM (post-translational modification), PD (Parkinson’s Disease) Declarations Competing Interests The authors report no competing interests. Author Contribution AA led the study, performed the experimental work, conducted data analyses and drafted the manuscript. AA provided daily mentorship and scientific guidance. DC and HM assisted with data analysis and scientific interpretation. WD, PV, JDPA contributed conceptual input and critically revised the manuscript. All authors approved the final manuscript. Acknowledgement We extend our sincere gratitude to Prof. Ryosuke Takahashi, Prof. Hodaka Yamakado and the members of their lab for critically reviewing this manuscript and for providing the cryopreserved heterozygous A53T-SNCA BAC embryos. We gratefully acknowledge the assistance of In Vivo Systems Research Support Staff at Johnson & Johnson for their contributions to animal husbandry and welfare. We want to thank Dr. Louis de Muynck for identifying the variation in mutant SNCA copy number in the A53T-SNCA BAC line, Dr. Wouter Bruinzeel for providing the amygdala extracts from a human Parkinson’s disease patient and Dr. Wim Van der Elst for statistical advice. 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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-9236452","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":615077940,"identity":"51562d20-4ff6-4bbd-9dfe-471872bc67a8","order_by":0,"name":"Annelore Anthonissen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYJACZiCu7weRDDxgDkHA2AwiZjYzk6plwwGiFAMBf/vZ448LKu4wGx/nP/yBQeYOOwP74aMb8GmROJOX2DzjzDM2s8PMbBIMPM+YGXjS0m7gteZAjmEzb9thHpAWoF8OMzNI8Jjh1SJ//g1Yi4RxMzPzB6K0GNyA2GJgwAxUTJQWwxtvDGfznDmcIHGY2UwiAaiFjZBf5M7nGHzmqTicwN9/8PGHjz2Hk/nZDx/D730UkNjDkMxGvHIw+MFgR6KOUTAKRsEoGAEAALwDQ6E20QHwAAAAAElFTkSuQmCC","orcid":"","institution":"Johnson \u0026 Johnson","correspondingAuthor":true,"prefix":"","firstName":"Annelore","middleName":"","lastName":"Anthonissen","suffix":""},{"id":615077941,"identity":"18e62aa3-11ba-4e87-9549-736793949723","order_by":1,"name":"Wilhelmus Drinkenburg","email":"","orcid":"","institution":"University of Groningen","correspondingAuthor":false,"prefix":"","firstName":"Wilhelmus","middleName":"","lastName":"Drinkenburg","suffix":""},{"id":615077942,"identity":"f61d919e-e477-46d2-a5cf-61d7576471a7","order_by":2,"name":"Patrik Verstreken","email":"","orcid":"","institution":"VIB-KU Leuven Center for Neuroscience","correspondingAuthor":false,"prefix":"","firstName":"Patrik","middleName":"","lastName":"Verstreken","suffix":""},{"id":615077943,"identity":"74f8977d-71d3-4d09-8d14-52b617be451c","order_by":3,"name":"Dries Crauwels","email":"","orcid":"","institution":"Johnson \u0026 Johnson","correspondingAuthor":false,"prefix":"","firstName":"Dries","middleName":"","lastName":"Crauwels","suffix":""},{"id":615077944,"identity":"e4a6bf07-f981-4fde-ae66-f2273dc0bb78","order_by":4,"name":"Juan Diego Pita Almenar","email":"","orcid":"","institution":"Johnson \u0026 Johnson","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"Diego Pita","lastName":"Almenar","suffix":""},{"id":615077945,"identity":"77a447d8-4de2-405f-ab59-d09685fc7d32","order_by":5,"name":"Hervé Maurin","email":"","orcid":"","institution":"Johnson \u0026 Johnson","correspondingAuthor":false,"prefix":"","firstName":"Hervé","middleName":"","lastName":"Maurin","suffix":""},{"id":615077946,"identity":"c0b207ec-0e09-4e0f-a5bc-4224b3ff6fe7","order_by":6,"name":"Abdellah Ahnaou","email":"","orcid":"","institution":"Johnson \u0026 Johnson","correspondingAuthor":false,"prefix":"","firstName":"Abdellah","middleName":"","lastName":"Ahnaou","suffix":""}],"badges":[],"createdAt":"2026-03-26 16:23:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9236452/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9236452/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106255569,"identity":"05ae3448-0817-401a-b397-8c5ab8747471","added_by":"auto","created_at":"2026-04-06 18:44:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":114903,"visible":true,"origin":"","legend":"\u003cp\u003eCopy number variation of human \u003cem\u003eSNCA\u003c/em\u003e transgene elucidates low-copy and high-copy groups of heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC mice. Mutant human \u003cem\u003eSNCA\u003c/em\u003e DNA copies were calculated from dividing the concentration [copies/µL] of the human \u003cem\u003eSNCA\u003c/em\u003e target gene by the concentration [copies/µL] of the mouse \u003cem\u003eAp3bi\u003c/em\u003e reference gene, multiplied by two for diploidy. Median of 32.76 was used as cut-off value to divide groups based on copy number. Low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice\u003cem\u003e \u003c/em\u003e(blue, n = 13) were defined by harboring less than 32.76 human \u003cem\u003eSNCA \u003c/em\u003ecopies, while high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice\u003cem\u003e \u003c/em\u003e(red, n = 7) were defined by having more than 32.76 human \u003cem\u003eSNCA \u003c/em\u003ecopies. **** (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001), unpaired Student’s \u003cem\u003et\u003c/em\u003e test.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/48c49aed6835571d6936b7f4.jpg"},{"id":106723985,"identity":"1fc29bcd-036f-4f86-a93a-ea43641694ef","added_by":"auto","created_at":"2026-04-12 18:23:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":245432,"visible":true,"origin":"","legend":"\u003cp\u003eSerine129 phosphorylated and C-terminally truncated α-Syn monomers are overexpressed in a \u003cem\u003eSNCA \u003c/em\u003etransgene dose-dependent manner without detectable aggregation in 24-month-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eBAC mice. (a) EP1536Y staining shows \u003cem\u003eSNCA\u003c/em\u003e dose-dependent increase in pS129-α-Syn in olfactory bulb, striatum and cortex; a 17kDa band in brainstem is likely due to non-specific binding. Human Parkinson’s disease brain sample, showing bands at monomeric level [~15kDa] and HMW level [≥198kDa], was used as positive control. (b) MJFR13-81A immunoassay does not detect HMW pS129-α-Syn in pooled olfactory bulb, striatum and cortex from \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice. Raw signals were converted to concentrations [ng/mL] by interpolation from the pS129-α-Syn PFF standard curve, showing a dose-dependent response to pS129-α-Syn PFF (dark), whereas values for wt (grey) and\u003cem\u003e SNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/- \u003c/em\u003e\u003c/sup\u003e(red) samples are at or below the limit of quantification (LOQ). Brain homogenates were pooled by genotype: 16 wild-type and 17 \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice. (c) Proteinase K treatment eliminates pS129-α-Syn monomers in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice, but not those observed in human Parkinson’s disease brain sample. (d) MJFR14 staining reveals \u003cem\u003eSNCA \u003c/em\u003edose-dependent increase in monomeric α-Syn [~15kDa] across regions, with no HMW α-Syn [≥198kDa] detected in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice; human Parkinson’s disease brain sample shows a diffused smear from ≥198kDa, indicative of mature fibrillar aggregates. Bands at ~14kDa in α-Syn knock-out samples originated from β-Synuclein. (e) Syn-1 staining confirms \u003cem\u003eSNCA \u003c/em\u003edose-dependent increases in monomeric α-Syn [~14kDa] and identifies C-terminally truncated α-Syn species [\u0026gt;14kDa] in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eBAC mice, particularly in olfactory bulb, striatum and cortex. Band intensities were normalized to β-actin and expressed as fold-change relative to wild-type controls. Green arrowheads mark specific antibody binding; orange arrowheads indicate non-specific binding in α-Syn knock-out samples Abbreviations: OB = olfactory bulb, STR = striatum, BS = brainstem, CTX = cortex, PD = Parkinson’s Disease, PFF = α-Syn preformed fibrils, PBS = phosphate buffered saline, wt = wild-type, PK = proteinase K. All original membranes are shown in Supplementary Figure 4. Each group is represented by a single animal (\u003cem\u003en \u003c/em\u003e= 1); inset bar charts illustrate expected α-Syn levels for each condition, with low \u003cem\u003eSNCA \u003c/em\u003egroup represented by one \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emale harboring 19.7 \u003cem\u003eSNCA \u003c/em\u003ecopies and high \u003cem\u003eSNCA \u003c/em\u003egroup represented by one \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emale harboring 56.6 \u003cem\u003eSNCA \u003c/em\u003ecopies. Accordingly, the results are descriptive with no inferential statistics.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/15888253fb1225ec39b04af6.jpg"},{"id":106255568,"identity":"b871f7f5-b54e-4e1b-a18e-dd7caad0984b","added_by":"auto","created_at":"2026-04-06 18:44:42","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":367916,"visible":true,"origin":"","legend":"\u003cp\u003eOlfactory assessments did not show early signs of robust olfactory impairments in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eBAC mice. (a) Latency \u0026nbsp;to locate a buried vanilla cookie did not differ between genotypes at 10 or 24 months. 10 months: wt (grey, \u003cem\u003en\u003c/em\u003e = 20), low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(blue, \u003cem\u003en\u003c/em\u003e = 14), high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(red, \u003cem\u003en\u003c/em\u003e = 6). 24 months: wt (grey, \u003cem\u003en\u003c/em\u003e = 15), low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(blue, \u003cem\u003en \u003c/em\u003e= 14, high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(red, \u003cem\u003en \u003c/em\u003e= 4). Linear mixed-effects model: \u003cem\u003eF\u003c/em\u003e(2,37) = 0.7066, \u003cem\u003eP = \u003c/em\u003e0.4999. Tukey’s post-hoc comparisons were non-significant. Bars represent mean ± SEM. (b-g) Olfactory discrimination task in 14-month-old mice exposed for 2min to water and vanilla odor (1:100). (b) Difference in sniffing time did not differ between genotypes. One-way ANOVA: \u003cem\u003eF\u003c/em\u003e(2,35) = 0.8339, \u003cem\u003eP = \u003c/em\u003e0.4428. Dunnett’s post-hoc comparisons were non-significant. (c) Power spectral density (PSD; [0.5-100Hz]) of olfactory bulb LFP during discrimination, expressed as percentage of baseline. Lines represent mean PSD for wt (grey, \u003cem\u003en\u003c/em\u003e = 17), low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eBAC\u003cem\u003e \u003c/em\u003e(blue, \u003cem\u003en\u003c/em\u003e = 13) and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eBAC mice (red, \u003cem\u003en\u003c/em\u003e = 7). (d-g) Normalized mean power across theta (d), alpha (e), beta (f) and gamma (g) bands showed no genotype effects. Kruskal-Wallis tests; all \u003cem\u003eP \u003c/em\u003e\u0026gt; 0.05. (h-j) Light-dark test at 24 months revealed no genotype-dependent changes in anxiety-like behavior. (h) All groups spent more time in the dark than light zone. Repeated measures two-way ANOVA: Compartment effect: \u003cem\u003eF\u003c/em\u003e(1,27) = 174,1, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001 (****); Compartment x Genotype effect: \u003cem\u003eF\u003c/em\u003e(2,27) = 0.7494,\u0026nbsp; \u003cem\u003eP = \u003c/em\u003e0.4822. (i) Time spent in light compartment and (j) number of light-zone entries did not differ between genotypes. One-way ANOVA: (i) F(2,27) = 0.7493, \u003cem\u003eP = \u003c/em\u003e0.4823. Dunnett’s post-hoc comparisons were non-significant. (j) One-way ANOVA: \u003cem\u003eF\u003c/em\u003e(2,27) = 0.8478, \u003cem\u003eP = \u003c/em\u003e0.4394. Dunnett’s post-hoc comparisons were non-significant. Bars represent mean ± SEM. Across experiments, low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice harbored 21.84±1.17 \u003cem\u003eSNCA\u003c/em\u003e copies and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice 53.04±1.39\u003cem\u003e SNCA\u003c/em\u003e copies. wt (grey, \u003cem\u003en\u003c/em\u003e = 16), low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(blue, \u003cem\u003en\u003c/em\u003e = 10, high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(red, \u003cem\u003en\u003c/em\u003e = 4). (k) Representative pS129-α-Syn [EP1536Y] immunostaining of olfactory bulb from distinctive 18-month-old cohort shows \u003cem\u003eSNCA \u003c/em\u003edose-dependent increase in immunoreactivity, with and without proteinase K treatment. \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eBAC mice display pS129-α-Syn depositions in inner plexiform layer, granule cell layer and subependymal zone, absent in wild-type mice. I = anterior olfactory bulb; II = glomerular layer to \u0026nbsp;granular layers; III = subependymal zone. \u0026nbsp;\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/e55cdaa4b612d4c57f43e8df.jpg"},{"id":106402709,"identity":"6ce8fa48-4a17-471d-b251-3469e68f8046","added_by":"auto","created_at":"2026-04-08 09:12:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":316523,"visible":true,"origin":"","legend":"\u003cp\u003eTemporal analysis of EMG activity during REM sleep does not reveal RBD-like phenotype in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice.\u0026nbsp; (a-c) Total duration of awake (a: linear mixed-effects model: genotype-effect: \u003cem\u003eF\u003c/em\u003e(2,37) = 0.8382,\u0026nbsp; \u003cem\u003eP = \u003c/em\u003e0.4406; genotype x time-effect: \u003cem\u003eF\u003c/em\u003e(4,67) = 1.971,\u0026nbsp; \u003cem\u003eP = \u003c/em\u003e0.1089), non-REM sleep (b: linear mixed-effects model: genotype-effect: \u003cem\u003eF\u003c/em\u003e(2,37) = 0.0261, \u003cem\u003eP = \u003c/em\u003e0.9743; genotype x time-effect: \u003cem\u003eF\u003c/em\u003e(4,67) = 1.164,\u0026nbsp; \u003cem\u003eP = \u003c/em\u003e0.3345) and REM sleep (c: linear mixed-effects model: genotype-effect: \u003cem\u003eF\u003c/em\u003e(2,37) = 0.4744, \u003cem\u003eP = \u003c/em\u003e0.6260; genotype x time-effect: \u003cem\u003eF\u003c/em\u003e(4,67) = 0.6360,\u0026nbsp; \u003cem\u003eP = \u003c/em\u003e0.6386) over a 24h recording does not significantly differ across groups. (d-e) No significant increase in REM-associated muscle activity over time in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice compared to wild-type mice, assessed by automated calculation of REM epochs with EMG\u003csub\u003eREM\u003c/sub\u003e power exceeding three standard deviations of EMG\u003csub\u003eNREM\u003c/sub\u003e power (d: linear mixed-effects model: genotype-effect: \u003cem\u003eF\u003c/em\u003e(2,37) = 1.392, \u003cem\u003eP\u0026nbsp; \u003c/em\u003e= 0.6571; genotype x time-effect: \u003cem\u003eF\u003c/em\u003e(4,64) = 0.6097, \u003cem\u003eP \u003c/em\u003e= 0.6571), and by manual counting REM epochs with elevated EMG amplitude and visible movement \u0026nbsp;(e: linear mixed-effects model: genotype-effect: \u003cem\u003eF\u003c/em\u003e(2,36) = 2.080, \u003cem\u003eP\u0026nbsp; \u003c/em\u003e= 0.1396; genotype x time-effect: \u003cem\u003eF\u003c/em\u003e(4,64) = 2.295, \u003cem\u003eP \u003c/em\u003e= 0.0755). wt (grey, \u003cem\u003en\u003c/em\u003e = 20), low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003eA53T/-\u003c/sup\u003e harboring 21.84±1.17 \u003cem\u003eSNCA\u003c/em\u003e copies (blue, \u003cem\u003en\u003c/em\u003e = 13), high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003eA53T/-\u0026nbsp; \u003c/sup\u003eharboring 53.04±1.39 \u003cem\u003eSNCA\u003c/em\u003e copies (red, \u003cem\u003en\u003c/em\u003e = 6). Vertical bars represent mean ± SEM. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 (*), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 (**), \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005 (***). (f) Frontal (FR) and parietal (PA) EEG and EMG traces reveal comparable REM-associated movements in 18-month-old wild-type, low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eBAC mice. (g) Representative \u0026nbsp;images of sublaterodorsal tegmental nucleus in brainstem of distinctive 18-month-old cohort of wt mice (\u003cem\u003en\u003c/em\u003e = 13) , low \u003cem\u003eSCNA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice (\u003cem\u003eSNCA \u003c/em\u003ecopies: 27.7, \u003cem\u003en \u003c/em\u003e= 10) and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003emice (\u003cem\u003eSNCA \u003c/em\u003ecopies: 57.2, \u003cem\u003en\u003c/em\u003e = 3) do not reveal any pS129-α-Syn [EP1536Y] deposition.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/cd5da169cee7d94aaae88928.jpg"},{"id":106402756,"identity":"0d1a322e-edf6-44d5-a374-10bdad132327","added_by":"auto","created_at":"2026-04-08 09:12:46","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":247574,"visible":true,"origin":"","legend":"\u003cp\u003eMotor assessment and power spectral density reveal preserved motor performance in 24-month-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice. (a-c) At 24 months, Catwalk XT gait analysis revealed no genotype-dependent differences in run speed (a: one-way ANOVA: \u003cem\u003eF\u003c/em\u003e(2,26) = 1.681, \u003cem\u003eP = \u003c/em\u003e0.2058), swing time (b: two-way ANOVA: \u003cem\u003eF\u003c/em\u003e(2,27) = 0.3125, \u003cem\u003eP = \u003c/em\u003e0.7342; genotype x paw-effect: \u003cem\u003eF\u003c/em\u003e(6,81) = 0.2418,\u0026nbsp; \u003cem\u003eP = \u003c/em\u003e0.9613) and stride length (c: two-way ANOVA: genotype-effect: \u003cem\u003eF\u003c/em\u003e(2,27) = 0.6377, \u003cem\u003eP = \u003c/em\u003e0.5363; genotype x raw-effect: \u003cem\u003eF\u003c/em\u003e(6,81) = 0.5096,\u0026nbsp; \u003cem\u003eP = \u003c/em\u003e0.7994) in wt (grey, \u003cem\u003en\u003c/em\u003e = 15), low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e (blue, \u003cem\u003en\u003c/em\u003e = 10) and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice (red, \u003cem\u003en\u003c/em\u003e = 4). (d) Rotarod performance over time showed no reduction in latency to fall in low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e (blue, \u003cem\u003en\u003c/em\u003e = 13) and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice (red, \u003cem\u003en \u003c/em\u003e= 7) compared to wt controls (grey, \u003cem\u003en\u003c/em\u003e = \u0026nbsp;21). Linear mixed-effects model: genotype-effect: \u003cem\u003eF\u003c/em\u003e(2,38) = 0.5212, \u003cem\u003eP = \u003c/em\u003e0.5980; genotype x time-effect: \u003cem\u003eF\u003c/em\u003e(12,209) = 1.383, \u003cem\u003eP = \u003c/em\u003e0.1758. (e-n) PSD [0.5-100Hz] of LFP recordings from primary motor cortex (M1; e) and substantia nigra (SN; j) during active wakefulness over 24h in 18-month-old mice. Lines represent mean PSD of wt (grey, \u003cem\u003en\u003c/em\u003e = 18), low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(blue, \u003cem\u003en \u003c/em\u003e= 13), and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e\u0026nbsp;mice (red, \u003cem\u003en\u003c/em\u003e = 7). (f-i, k-n) Normalized mean power across theta, alpha, beta and gamma frequency bands did not differ between genotypes in either region. One-way ANOVA: all \u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05. Dunnett’s post-hoc comparisons were non-significant. Across experiments, low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice harbored 21.84±1.17 human \u003cem\u003eSNCA\u003c/em\u003e copies and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003emice 53.04±1.39 human\u003cem\u003e SNCA\u003c/em\u003e copies. (o) Representative pS129-α-Syn [EP1536Y] immnostaining of striatum from distinctive 18-month-old cohort shows \u003cem\u003eSNCA-\u003c/em\u003edose dependent deposition of sparse extracellular or neuritic pS129-α-Syn in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eBAC mice. wt (\u003cem\u003en\u003c/em\u003e = 13), low \u003cem\u003eSCNA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u0026nbsp;(\u003cem\u003eSNCA \u003c/em\u003ecopies: 27.7, \u003cem\u003en\u003c/em\u003e = 9) and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice (\u003cem\u003eSNCA \u003c/em\u003ecopies: 57.7, \u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/c0e4087b3426d7794e63bc5c.jpg"},{"id":106255572,"identity":"23d05742-d02a-4cbe-916e-4aa4ba0ad195","added_by":"auto","created_at":"2026-04-06 18:44:42","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":247148,"visible":true,"origin":"","legend":"\u003cp\u003eTyrosine hydroxylase immunoreactivity does not reveal dopaminergic neurodegeneration along striatum, nigrostriatal pathway and substantia nigra in 24-month-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eBAC mice. Representative tyrosine hydroxylase (TH) staining of stiatum (STR), nigrostriatal pathway (NSP) and substantia nigra \u003cem\u003epars compacta \u003c/em\u003e(SN \u003cem\u003epc\u003c/em\u003e) of \u0026nbsp;24-month-old wild-type, low \u003cem\u003eSCNA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e\u0026nbsp;(\u003cem\u003eSNCA \u003c/em\u003ecopies: 20) and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice (\u003cem\u003eSNCA \u003c/em\u003ecopies: 54.2) did not show a decrease in TH mean intensity and ratio of TH area/region of interest in low (n = 10) and high (\u003cem\u003en\u003c/em\u003e = 6) \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice, compared to wild-type mice (\u003cem\u003en \u003c/em\u003e= 12) at 24 months of age. Ordinary one-way ANOVA with Dunnet’s post hoc comparisons. TH mean intensity: (STR) \u003cem\u003eF\u003c/em\u003e(2,22) = 0.1516, \u003cem\u003eP\u003c/em\u003e = 0.8602; (NSP) \u003cem\u003eF\u003c/em\u003e(2,22) = 0.4904, \u003cem\u003eP\u003c/em\u003e = 0.6189; (SN\u003cem\u003epc\u003c/em\u003e) \u003cem\u003eF\u003c/em\u003e(2,22) = \u0026nbsp;0.5434, \u003cem\u003eP\u003c/em\u003e = 0.5884. Ratio TH area/region of interest: (STR) \u003cem\u003eF\u003c/em\u003e(2,22) = 0.2839, \u003cem\u003eP \u003c/em\u003e= 0.7556; (NSP) \u003cem\u003eF\u003c/em\u003e(2,22) = 0.7151, \u003cem\u003eP\u003c/em\u003e = 0.5002; (SN\u003cem\u003epc\u003c/em\u003e) \u003cem\u003eF\u003c/em\u003e(2,22) = 1.112, \u0026nbsp;\u003cem\u003eP\u003c/em\u003e = 0.3468.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/ef986af55e3692198cac07da.jpg"},{"id":106725449,"identity":"93ce3429-49e3-4175-a915-d9efceae071b","added_by":"auto","created_at":"2026-04-12 18:32:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2650494,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/e62bbb30-1049-4cf3-98ee-70e9c02bb2de.pdf"},{"id":106255567,"identity":"78a99752-3e46-4d53-8473-c19dbf1a105c","added_by":"auto","created_at":"2026-04-06 18:44:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":5233957,"visible":true,"origin":"","legend":"","description":"","filename":"AnthonissenASupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-9236452/v1/a3859ca68a2e4a5e0693dade.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phosphorylation and Truncation of α-Synuclein do not trigger Parkinsonian Readouts in A53T-SNCA Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease affects approximately 1% of the global population over 60 years of age, making it the second most common neurodegenerative disorder. Pathologically, it is characterized by the aggregation of misfolded intraneuronal α-Synuclein (α-Syn) deposits termed \u0026lsquo;Lewy Bodies\u0026rsquo; and nigrostriatal dopaminergic neurodegeneration. These result in the progressive manifestation of motor impairments including bradykinesia, postural instability, resting tremor and rigidity.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAlthough clinical diagnosis focuses on motor impairments, growing evidence emphasizes the importance of non-motor symptoms that emerge years before motor dysfunctions appear in patients.\u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e This pre-motor stage may offer a therapeutic window to test new disease-modifying approaches intended to modify or halt the progression of the disease early on. Additionally, the use of prodromal biomarkers may advance early identification of people at risk for developing Parkinson\u0026rsquo;s disease. This will attribute to a more accurate delineation of target populations for neuroprotective trials, facilitating the development of precision medicine-based therapies.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRecent evidence from multicenter studies indicates olfactory dysfunction and rapid eye movement sleep behavior disorder (RBD) as strong predictors of phenoconversion to parkinsonism, including Parkinson\u0026rsquo;s Disease, Dementia with Lewy Bodies and Multiple System Atrophy. The high prevalence of olfactory dysfunction and RBD in Parkinson\u0026rsquo;s disease, along with Braak\u0026rsquo;s hypothesis stating that the pathology initially targets the olfactory bulb and brainstem, supports their potential as predictive biomarkers.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRoutinely used animal models do not replicate the progressive multisystem nature of the disorder, including its early non-motor phase and pathology beyond the nigrostriatal pathway. Focusing on models that reproduce the spatiotemporal prodromal-to-late progression of Parkinson\u0026rsquo;s disease may enhance insights into the pathogenesis and strengthen translational validity.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eTherefore, we evaluated a prodromal Parkinson\u0026rsquo;s disease mouse model described by Taguchi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e that harbors a bacterial artificial chromosome (BAC) with the human \u003cem\u003eSNCA\u003c/em\u003e gene in which the \u003cem\u003eA53T\u003c/em\u003e mutation, two risk-associated single-nucleotide polymorphisms (rs11931074, rs3857059) and a Rep1 dinucleotide repeat polymorphism were introduced. According to their study, this model exhibits α-Syn pathology across multiple brain regions, dopaminergic neurodegeneration, early signs of olfactory dysfunction and RBD, but no motor impairments at the age of 18 months.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn our study, we investigated whether \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent overexpression of α-Syn, including Serine129 phosphorylated and C-terminally truncated forms, was sufficient to cause key pathological and functional readouts featuring prodromal-to-late Parkinson\u0026rsquo;s disease in heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC mice (\u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC) over a 24-month period.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eCopy number variation in heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC Mice\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eTo ensure genetic homogeneity, we quantified human \u003cem\u003eSNCA\u003c/em\u003e copies in our longitudinal cohort of 20 \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC mice using ddPCR \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. \u003cem\u003eSNCA\u003c/em\u003e copies ranged from 17.8 to 58.5 (median 32.76), and mice were therefore divided into low (\u0026lt;\u0026thinsp;32.76 \u003cem\u003eSNCA\u003c/em\u003e copies) and high (\u0026gt;\u0026thinsp;32.76 \u003cem\u003eSNCA\u003c/em\u003e copies) \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e groups. The average copy number of human \u003cem\u003eSNCA\u003c/em\u003e in low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e group (21.84\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17) was significantly lower (difference: 31.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e group (53.04\u0026thinsp;\u0026plusmn;\u0026thinsp;1.39). The stratification of the transgenic group allowed us to assess whether variability in \u003cem\u003eSNCA\u003c/em\u003e transgene dose impacted α-Syn expression and the onset or severity of functional readouts related to Parkinson\u0026rsquo;s disase.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eOverexpression of pS129 and truncated α-Syn do not induce fibrillar aggregation in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eTo assess α-Syn pathology in 24-month-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice, we performed SDS-PAGE Western blotting on pooled brain homogenates using pS129-α-Syn (EP1536Y) and α-Syn (MJFR14 \u0026amp; Syn-1) antibodies. pS129-α-Syn monomers [~\u0026thinsp;15kDa] increased in \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent manner in olfactory bulb, striatum and cortex, with the highest levels in the olfactory bulb, while no increase was detected in brainstem (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). In contrast to the human Parkinson\u0026rsquo;s disease brain sample, no comparable higher-molecular-weight (HMW) pS129-α-Syn species [\u0026ge;\u0026thinsp;198kDa] were observed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e. This was confirmed by the MJFR13-81A immunoassay, in which interpolated values for pooled olfactory bulb, striatum and cortex homogenates from \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice fell at or below the limit of quantification \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Additionally, proteinase K treatment eliminated monomeric pS129-α-Syn in all regions of \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice, whereas both monomeric and HMW α-Syn persisted in the human Parkinson\u0026rsquo;s disease brain sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), indicating that pathological α-Syn species associated with Parkinson\u0026rsquo;s disease are proteinase K-resistant.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the presence of pathological α-Syn aggregates in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice, MJFR14 solely revealed a \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent increase in monomeric α-Syn levels [~\u0026thinsp;15kDa] across all regions. Given the denaturing conditions of SDS-PAGE, monomeric α-Syn signals could theoretically arise from dissociation of fragile, non-covalent early oligomeric forms. Nevertheless, the SDS-resistant HMW α-Syn aggregates [\u0026ge;\u0026thinsp;198kDa] observed in human Parkinson\u0026rsquo;s disease brain sample were not detected in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e. Furthermore, Syn-1 showed a \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent rise in monomeric α-Syn [~\u0026thinsp;15kDa] across all brain regions and additionally revealed C-terminally truncated α-Syn species [\u0026lt;\u0026thinsp;15kDa] in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice, most abundant in olfactory bulb, striatum and cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003eOverall, \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice displayed region- and \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent elevations in pS129 and C-terminally truncated α-Syn monomers, but no evidence of SDS-resistant HMW α-Syn species [\u0026ge;\u0026thinsp;198kDa] comparable to those detected in the human Parkinson\u0026rsquo;s disease brain sample at 24 months.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eNo signs of olfactory dysfunction in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eOlfactory dysfunction may signal underlying α-Syn pathology many years before a clinical diagnosis of Parkinson\u0026rsquo;s disease.\u003csup\u003e\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Therefore, we evaluated whether \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice exhibit impairments in olfactory detection and discrimination. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, the buried food seeking test performed at 10 and 24 months showed no disability in olfactory detection in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice compared to controls. Additionally, olfactory discrimination \u0026ndash; measured as the difference in sniffing time between water and vanilla - was not significantly altered in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice compared with controls at 14 months \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. Moreover, LFP of the olfactory bulb recorded during the discrimination paradigm showed no significant deviations in power spectral density between genotypes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e across physiologically relevant frequency bands: theta [4-8Hz] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e, alpha [8-12Hz] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e, beta [13-30Hz] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef\u003cb\u003e)\u003c/b\u003e and gamma [30-100Hz] \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e. Given that olfactory performance can be affected by various confounding factors such as anxiety or depression, we performed the light-dark test. Cumulative duration spent in both compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh\u003cb\u003e)\u003c/b\u003e, time in light compartment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei\u003cb\u003e)\u003c/b\u003e and number of entries to light compartment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej\u003cb\u003e)\u003c/b\u003e revealed no differences between \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice and controls. Hence, olfactory performance was likely not affected by differences in anxiety levels.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we assessed pS129-α-Syn levels and found significant \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent labeling in the olfactory bulbs of 18-month-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice compared to controls \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek\u003cb\u003e)\u003c/b\u003e. This immunoreactivity appeared to persist over time, as a separate cohort of 4-month-old wild-type and \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice showed similar pattern and intensity of the immunoreactivity \u003cb\u003e(Supplementary Fig.\u0026nbsp;5)\u003c/b\u003e. In wild-type mice, low physiological levels of pS129-α-Syn are found in the anterior olfactory bulb, mitral cell layer, external plexiform layer and glomerular layer. In contrast, \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice exhibit higher intensity of pS129-α-Syn in these regions, along with immunoreactivity in the inner plexiform layer, granule cell layer and subependymal zone. Interestingly, upon incubation with proteinase K, no pS129-α-Syn was detected in wild-type mice, whereas \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent immunoreactivity in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice persisted in regions that normally contain physiological pS129-α-Syn, but disappeared from the inner plexiform layer, granule cell layer and subependymal zone. However, the interpretation that this reflects the presence of PK-resistant pS129-α-Syn species should be made cautiously, as suboptimal proteolysis at the employed PK dose could result in residual immunoreactivity. This caution is supported by western blot data \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, showing that pS129-α-Syn in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC homogenates was fully degraded under PK conditions.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eNo early REM sleep behavior disorder-like phenotype observed in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eConsidering the emerging link between prodromal RBD and the phenoconversion to Parkinson\u0026rsquo;s disease\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, we performed a longitudinal assessment of motor activity during REM sleep in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice. At various time points, we assessed their 24-hour sleep-wake architecture using EEG-EMG recordings, complemented by passive infrared monitoring and synchronized video tracking. \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice showed no significant differences in total wake, NREM or REM sleep duration compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea\u0026ndash;c). This indicates that subsequent analyses of RBD-like behavior are unlikely to be influenced by REM duration. Since RBD is characterized by REM sleep without atonia (RSWA), we examined whether \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice exhibited increased muscle activity during REM sleep. We used two calculation approaches: (1) an automated detection of REM epochs with EMG\u003csub\u003eREM\u003c/sub\u003e power exceeding three standard deviations of EMG\u003csub\u003eNREM\u003c/sub\u003e power \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ed\u003cb\u003e)\u003c/b\u003e and (2) a manual screening of REM epochs with elevated EMG amplitude and visible movement \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e. Neither approach showed a significant increase in muscle activity in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice, irrespective of their transgene dose, compared to wild-type mice at any time point. Additionally, EEG-EMG traces showed comparable REM-associated movements across wild-type, low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e and high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). This suggests that the movements in this study may not be considered abnormal events featuring an RBD-like phenotype but rather represent normal biological phenomena during REM sleep. Collectively, the lack of increased EMG activity indicates that \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice do not exhibit RBD-like behavior. Furthermore, the sublaterodorsal tegmental nucleus, a region in the brainstem that regulates muscle tone during sleep-wake cycles,\u003csup\u003e28\u0026ndash;30\u003c/sup\u003e showed no deposition of pS129-α-Syn in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice, potentially corroborating with the absence of RBD-like features \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eg\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003ePreserved motor performance in 24-month-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe main pathological feature of Parkinson\u0026rsquo;s disease involves the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. These neurons project through the nigrostriatal pathway to the striatum resulting in a striatal depletion of dopamine that dysregulates motor activity.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Consequently, bradykinesia, rigidity, postural instability and resting tremor are considered cardinal signs of Parkinson\u0026rsquo;s disease.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e To assess progressive motor dysfunctions in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice, we conducted the Rotarod motor performance test and Catwalk XT gait analysis at multiple timepoints up to 24 months. Temporal and spatial gait parameters showed no significant deviations between \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC and wild-type mice at 24 months (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-c). Additionally, latency to fall in the accelerating Rotarod test did not differ between \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice and wild-type mice \u003cb\u003e(Fig.\u0026nbsp;(5d)\u003c/b\u003e. Since no motoric readouts were detectable yet, we evaluated whether neurophysiological alterations might occur in these mice, potentially preceding a future overt motor dysfunction. Therefore, we analyzed LFPs recorded in primary motor cortex \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e and substantia nigra \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ej\u003cb\u003e)\u003c/b\u003e during active wake stages over a 24-hour period. At 18 months, LFPs in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice revealed no significant changes in mean power across different physiological frequency bands, including theta [4-8Hz], alpha [8-12Hz], beta [13-30Hz] and gamma [30-100Hz], compared to wild-type mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-i \u003cb\u003e\u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ek-n\u003cb\u003e)\u003c/b\u003e. Immunohistochemical analysis of striatum in a distinctive cohort of 18-month-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice revealed \u003cem\u003eSNCA\u003c/em\u003e transgene dose\u003cem\u003e-\u003c/em\u003edependent extracellular and neuritic pS129-α-Syn depositions compared to wild-type mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eo\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess dopaminergic neurodegeneration, we evaluated the survival of tyrosine hydroxylase-positive neurons in the striatum, nigrostriatal pathway and substantia nigra of \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice after 24 months. Similar intensity averages were observed across all groups, as well as an equal ratio of immunoreactive area over the regions of interest (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This indicates that \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice in this study do not exhibit robust dopaminergic neurodegeneration after two years, despite the presence of monomeric pS129 and C-terminally truncated α-Syn in the striatum and midbrain as observed through immunohistochemistry \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eo\u003cb\u003e)\u003c/b\u003e and western blotting \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u003cb\u003e\u0026amp;\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we evaluated the translational validity of heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC mice as a model capturing prodromal-to-late Parkinson\u0026rsquo;s disease progression. This line carries the human \u003cem\u003eSNCA\u003c/em\u003e gene with the \u003cem\u003eA53T\u003c/em\u003e point mutation, two risk-associated single nucleotide polymorphisms and the Rep1 repeat.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e Although previous reports described Parkinsonian phenotypes in this model,\u003csup\u003e13,34,35\u003c/sup\u003e we did not observe such features under our experimental conditions. Despite copy number variation of the mutant \u003cem\u003eSNCA\u003c/em\u003e gene across the cohort, even high-copy mice remained free of non-motor and motor deficits and showed no electrophysiological abnormalities over a 24-month period. At the molecular level, \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent increases in pS129 and C-terminally truncated α-Syn species was observed in olfactory bulb, striatum and cortex; however, these changes were not accompanied by SDS-resistant HMW α-Syn species [\u0026ge;\u0026thinsp;198kDa], similar to those observed in Parkinson\u0026rsquo;s disease brain sample. Taken together, our findings suggest that the \u003cem\u003eA53T\u003c/em\u003e mutation together with additional risk-associated polymorphisms in human \u003cem\u003eSNCA\u003c/em\u003e gene are insufficient to drive the pathogenic processes underlying pre-motor or motor manifestations in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice within a 2-year window. The lack of mature fibrillar aggregates and Parkinsonian readouts further indicates that Ser129 phosphorylation and C-terminal truncation of α-Syn alone do not promote overt α-Syn aggregation \u003cem\u003ein vivo\u003c/em\u003e, or, alternatively, one could argue that the observed α-Syn overexpression does not surpass the threshold required for initiating pathogenic cascades.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eCopy number variation in heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC mice\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eWe observed a variability in copies of the mutant \u003cem\u003eSNCA\u003c/em\u003e gene in our heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC mice.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e This was surprising since all mice originated from a breeding pair with a heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC parent preselected for high mutant \u003cem\u003eSNCA\u003c/em\u003e dose. This underscores the importance of monitoring transgene copy number variation in BAC-based animal models.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e The copy number variation across our cohort is unlikely to result from random integration of the BAC construct, as a consistent 1:1 segregation ratio was maintained during breeding. Instead, this variability may reflect recombination of the large BAC construct during meiosis, resulting in diverse copy numbers or by the instability of the Rep1 dinucleotide repeat polymorphism that plays a pivotal role in the transcriptional regulation of the human \u003cem\u003eSNCA\u003c/em\u003e gene.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Future studies should focus on advancing BAC constructs to enable more targeted transgene delivery and to mitigate genetic drifts while maintaining sufficient \u003cem\u003eSNCA\u003c/em\u003e expression to induce pathological changes.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003epS129 and truncated α-Syn overexpression fails to induce fibrillar aggregation in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eOur study showed that the increase of mutant \u003cem\u003eSNCA\u003c/em\u003e copies was positively correlated with the overexpression of monomeric α-Syn and the abundance of Serine 129 phosphorylation and C-terminal truncation across various brain regions in 2-year-old \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice. It has been demonstrated that these PTMs alter the propensity of α-Syn to promote aggregation by affecting structure and conformation, and consequently its solubility.\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e However, within the context of our experiments, we did not detect HMW (pS129-) α-Syn species [\u0026ge;\u0026thinsp;198kDa] that would imply the presence of mature fibrillar (pS129-) α-Syn aggregates resembling those of the Parkinson\u0026rsquo;s disease brain sample. Possibly, mice with highest mutant \u003cem\u003eSNCA\u003c/em\u003e expression in this study may still have insufficient levels to induce α-Syn aggregation.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Another explanation, of a technical nature, could be the use of SDS-PAGE western blotting to detect HMW α-Syn species. Since SDS disrupts non-covalent interactions within aggregates, it could cause dissociation into smaller and more fragile fragments, masking the presence of early oligomeric species. In line with this, Taguchi \u003cem\u003eet al.\u003c/em\u003e reported the presence of oligomeric α-Syn using O1 immunostaining, but did not detect fibrillar aggregates with the fibril-specific F2 antibody\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, supporting the notion that α-Syn species in this model do not resemble the SDS-resistant fibrillar aggregates featuring Parkinson\u0026rsquo;s disease. To evaluate the potency of the present forms of pS129-α-Syn to promote aggregation in this model, we assessed their resistance to proteinase K. While pathogenic pS129-α-Syn species from the Parkinson\u0026rsquo;s disease brain sample remained resistant to proteinase K digestion due to their insolubility, all pS129-α-Syn were eliminated in the model. This suggests that pS129-α-Syn species in our mice are vulnerable for digestion due to their soluble nature, explaining their inability to induce fibrillar aggregation.\u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eInterestingly, our observations are in line with previous studies highlighting the physiological role of PTMs - Serine 129 phosphorylation and C-terminal truncation - and the lack of consensus on their pathogenic contribution to aggregation.\u003csup\u003e\u003cspan additionalcitationids=\"CR49 CR50 CR51 CR52\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e A recent study showed that after injection of α-Syn preformed fibrils into the striatum of mice, pS129-α-Syn became apparent four weeks post-injection, suggesting that phosphorylation occurs after the initial seeding and protein aggregation.\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e Additionally, \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice also demonstrated C-terminal truncation of α-Syn, which is believed to promote aggregation as it removes protective regions.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e However, the mice did not exhibit HMW α-Syn species, challenging its causative role in aggregation. Recent work by Mahul-Mellier \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e55\u003c/sup\u003e has shown that C-terminal truncation primarily acts as a post-fibrillization regulator that drives fibril remodeling and maturation into Lewy body\u0026ndash;like inclusions, rather than initiating aggregation. This suggests that Ser129 phosphorylation and C-terminal truncation could be epiphenomena that accompany aggregation, rather than playing direct causal roles in pathogenesis.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eLack of phenotypic readouts related to Parkinson\u0026rsquo;s disease in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e mice\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eBehavioral assessments with corresponding neurophysiological recordings revealed no early signs of REM sleep behavior disorder, olfactory dysfunction or late motor impairments in our \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/ \u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC cohort. Notably, this absence of prodromal non-motor symptoms contrasts with the findings reported by Taguchi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e Nevertheless, our functional readouts may be attributable to the absence of α-Syn aggregates in the relevant regions. Similarly, previous work by Moceri \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e35\u003c/sup\u003e showed no early olfactory defect in \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC mice, despite immunohistochemical analyses revealing high levels of pS129-α-Syn in the olfactory bulb. These findings strengthen the yet-unknown biological role of pS129-α-Syn in the olfactory bulb, where it is abundantly present in the healthy brains of both humans and mice.\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e A clinicopathological study by Tremblay \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e57\u003c/sup\u003e revealed that pS129-α-Syn deposition in the olfactory bulb of humans with age-related olfactory decline was not found to correlate with their olfactory performance, suggesting pS129-α-Syn does not seem to independently drive olfactory deficits. Nevertheless, a potential shortcoming of this study involves the use of conventional olfactory paradigms to evaluate olfactory performance. They provide a robust interpretation of whether mice can discriminate or perceive odors well but lack sensitivity to more subtle deficits. Therefore, future studies may benefit from using an automated olfactometer that standardizes olfactory stimulation by controlling odor concentrations, release time and flow rate.\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFurthermore, the absence of increased muscle activity during REM sleep in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice may reflect a lack of pS129-α-Syn deposition in the sublaterodorsal tegmental nucleus, a region implicated in the pathogenesis of RBD in prodromal Parkinson\u0026rsquo;s disease.\u003csup\u003e\u003cspan additionalcitationids=\"CR61 CR62\" citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e Interestingly, Shen \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e28\u003c/sup\u003e demonstrated that inoculating α-Syn fibrils into the SLD of mice triggers RBD-like behavior and later Parkinson\u0026rsquo;s disease, supporting a pathogenic role for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\alpha\\:\\)\u003c/span\u003e\u003c/span\u003e-Syn pathology in RBD. Nevertheless, characterizing RBD-like behavior in mice requires standardization, as the current diversity of assessment methods hinder reproducibility and reliable interpretation. Existing studies often infer RBD-like phenotypes from automated EEG-EMG quantification.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e Although these approaches provide an indication of muscle tone, they should be supplemented by synchronized videos to distinguish pathological phenomena from physiological movements or potential artefacts during REM sleep. In this study, the EEG-EMG traces with synchronized video show that the movements during REM sleep in \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice are identical to those in wild-type controls, indicating that these events are physiological rather than genotype-specific RSWA-like events. Standardized protocols combining EEG\u0026ndash;EMG, synchronized video and non-transgenic controls are therefore essential to avoid misclassification and to determine whether mice can truly exhibit RBD-like behavior.\u003c/p\u003e \u003cp\u003eTo be translationally relevant for Parkinson\u0026rsquo;s disease, a mouse model must exhibit nigrostriatal dopaminergic neurodegeneration and motor impairments. In this study, \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice did not exhibit a reduction in TH-immunoreactivity \u0026ndash; marker for dopaminergic neurons - along the nigrostriatal system at 24 months. Notably, the subtle reduction in TH-positive neurons reported by Taguchi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e at 18 months was identified using a more spatially resolved analysis across the entire midbrain, which may explain the discrepancy with our present findings. Additionally, these mice showed no evidence of altered neuronal activity in the substantia nigra or impaired motor performance in the CatWalk and RotaRod assessments. These findings corroborate those of Taguchi \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e13\u003c/sup\u003e and confirm that aging beyond 18 months does not lead to progressive motor impairments as previously hypothesized. Previous studies using transgenic mice overexpressing human α-Syn have similarly reported neither locomotor deficits nor clear dopaminergic neurodegeneration, despite substantial accumulation of (pS129-) α-Syn across multiple brain regions.\u003csup\u003e\u003cspan additionalcitationids=\"CR66\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e Together, these findings highlight the need to identify additional mechanisms that drive late Parkinsonian motor dysfunction in mouse models.\u003c/p\u003e \u003cp\u003eIn this 24-month study of heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e BAC mice, we assessed α-Syn pathology, neural activity and behaviors relevant to both prodromal and advanced Parkinson\u0026rsquo;s disease. We observed \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent overexpression of Serine129 phosphorylated and C-terminally truncated α-Syn across the olfactory bulb, striatum and cortex. Despite these molecular alterations, we did not detect mature fibrillar α-Syn aggregation, prodromal features including olfactory impairments or RBD-like manifestations, nor a progression to pronounced dopaminergic neurodegeneration or motor deficits. Collectively, these findings indicate that overexpression of pS129 and C-terminally truncated α-Syn alone is insufficient to drive aggregation or functional readouts associated with Parkinson\u0026rsquo;s disease within 24 months. This highlights the need to identify additional factors required to recapitulate prodromal-to-late motoric Parkinson\u0026rsquo;s disease in mouse models.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAnimal Husbandry\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAnimal housing, handling and experiments were strictly in accordance with the international directives of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by the Ethical Committee for Animal Experimentation at Johnson \u0026amp; Johnson. ARRIVE guidelines were used for reporting \u003cem\u003ein vivo\u003c/em\u003e research.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eAnimals were housed on a 12h dark/light cycle in Emerald EM500 cages with enrichment, food (SAFE\u0026reg; A05) and water access \u003cem\u003ead libitum\u003c/em\u003e, except for fasting experiments. Behavioral cohorts were single housed; molecular cohorts were group housed. Environmental conditions such as temperature, humidity and pressure were automatically controlled to maintain respective levels of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10% and 4 Pa. All behavioral experiments were conducted in the dark to respect mice\u0026rsquo;s nocturnal activity.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAnimal Breeding \u0026amp; Genotyping\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eCryopreserved \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC embryos (N10) with high \u003cem\u003eSNCA\u003c/em\u003e transgene dose were kindly provided by Prof. Dr. Hodaka Yamakado (Kyoto University).\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e \u003cem\u003eSCNA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/-\u003c/em\u003e\u003c/sup\u003e BAC males and wild-type C57BL/6J females were paired at the Transgenic Rodent Facility of Johnson \u0026amp; Johnson, and N15 offspring were used for experiments. Genotyping was performed on tail biopsies using the Bio-Rad QX200 Droplet Digital Polymerase Chain Reaction (ddPCR) system (IGBMC) to assess \u003cem\u003eA53T-SNCA\u003c/em\u003e copy number variation. Genomic DNA was extracted with the DNeasy Blood \u0026amp; Tissue Kit (Qiagen) and quantified via NanodropTM spectrophotometer (ThermoFisher Scientific). ddPCR reactions contained 10ng gDNA, 2X ddPCR supermix, 20X TaqMan probes for human \u003cem\u003eSNCA\u003c/em\u003e and mouse \u003cem\u003eAp3b1\u003c/em\u003e and nuclease-free water (InvitrogenTM). Droplets were generated, read on the QX200 system and analyzed with QuantaSoft Analysis Pro. \u003cem\u003eSNCA\u003c/em\u003e copy number was calculated as the \u003cem\u003eSNCA\u003c/em\u003e/\u003cem\u003eAp3b1\u003c/em\u003e ratio and multiplied for diploidy.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eAnimal Cohorts\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eLongitudinal assessments - including behavioral testing, EEG-EMG and local field potential (LFP) recordings - were performed on wild-type C57BL/6J mice (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;21) and \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e BAC mice (\u003cem\u003en\u0026thinsp;=\u003c/em\u003e\u0026thinsp;20), which were divided into low-copy (low \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13) and high-copy (high \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7) groups. At 24 months, mice were sacrificed, with the left hemisphere used for immunohistochemistry and the right hemisphere for western blotting and 2-step direct immunoassay. Additional cohorts were aged to 4 months (wt: \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8; \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8) and 18 months (wt: \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8; \u003cem\u003eSNCA\u003c/em\u003e\u003csup\u003e\u003cem\u003eA53T/\u0026minus;\u003c/em\u003e\u003c/sup\u003e: \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8) for age-dependent pathology mapping by immunohistochemistry. Male and female outcomes were analyzed separately but reported together, as no significant gender differences were observed. \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e illustrates the experimental design of this study.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eStereotaxic Electrode Implantation\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eSurgeries were performed on 2-month-old mice after induction with 5% isoflurane anesthesia, with morphine (2mg/kg) and Metacam\u0026reg; (5mg/kg) for analgesia. Isobetadine (Meda Pharma SA) and 70% Ethanol was applied to disinfect the surgical site, and eye ointment (Opticorn A, EcuPhar BV) was used to prevent dehydration during surgery. Once mice were fixed in the stereotaxic frame, anesthesia was maintained by 2%-2.5% isoflurane and body temperature was regulated by Harvard homeothermic monitoring system.\u003c/p\u003e \u003cp\u003eTwo stainless-steel EEG electrodes (7N51465T5TLT, 51/46 Teflon Bilaney) were implanted over the left frontal cortex (AP\u0026thinsp;+\u0026thinsp;2.3mm, ML -1.5mm) and parietal cortex (AP -1.5mm, ML -1mm). A reference electrode was placed 1mm posterior to lambda, and a ground electrode was positioned centrally to reduce noise. Four polyamide-coated stainless-steel depth electrodes (100\u0026micro;m diameter with a blunt-tip, customized by Peira bvba, Komax) were implanted in the right hemisphere at following coordinates (from dura): olfactory bulb (AP\u0026thinsp;+\u0026thinsp;4.5mm, +ML 1.2 mm, DV -0.8mm), primary motor cortex (AP\u0026thinsp;+\u0026thinsp;1.5mm, ML\u0026thinsp;+\u0026thinsp;1.6mm, DV -0.7mm), CA1 (AP -1.94mm, ML\u0026thinsp;+\u0026thinsp;1.2mm, DV -1.15mm) and substantia nigra (AP -3.28mm, ML\u0026thinsp;+\u0026thinsp;1.2mm, DV -4.3mm). Fine-wired electrodes were inserted in two trapezius muscles for EMG. The electrode configuration is shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e. All electrodes were secured with dental cement (Relyx Unicem 2 cement, 3M) and connected to a 10-channel headmount. Skin was closed with tissue-adhesive glue (Vetbond, 3M). Mice received 0.9% NaCl post-recovery, were closely monitored for 1 hour, and then housed in a heated cabinet with DietGel\u0026reg; 76A for 24 hours before daily checks for four days.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eVideo-assisted Electrophysiological Recording\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eAfter 2 weeks of recovery, mice were placed in sound-attenuated chambers with enrichment, food and water access \u003cem\u003ead libitum\u003c/em\u003e, and maintained under similar housing conditions. Each mouse underwent 48 hours of continuous recording, including a 24-hour habituation period, under a maintained light-dark cycle. Two video cameras (uEye CP, IDS Imaging GmbH) captured top and lateral views of behavior. EEG, LFP and EMG signals were acquired at 512Hz using the BioSemi system and stored in BDF format. General motor activity was also monitored via two passive infrared detectors placed above each cage.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eElectrophysiological Data Analysis\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e\n\u003ch3\u003eRaw Data Processing\u003c/h3\u003e\n\u003cp\u003eBDF files were imported into a LabVIEW-based analysis tool (National Instruments). Data were pre-processed with a 50Hz-Notch filter and a 1-200Hz band-pass Butterworth filter. Recordings were referenced to the reference electrode and grounded to reduce unwanted noise and interference from the environment. Visual inspection facilitated quality check and potential exclusion of signals showing flat lines indicating disconnection, high levels of artifacts, or widespread interference across electrodes.\u003c/p\u003e\n\u003ch3\u003eVigilance State Analysis\u003c/h3\u003e\n\u003cp\u003eSleep-wake states were scored in 2s-epochs over 24 hours of EEG-EMG-video recordings using a semi-automated LabVIEW-based tool (National Instruments). A machine learning algorithm provided initial scoring, followed by manual verification of the EEG-EMG traces and synchronized videos to accurately assign active wake, quiet wake, NREM sleep and REM sleep. Active wake was characterized by low-amplitude EEG with beta activity [13-30Hz], high EMG and PIR\u0026thinsp;\u0026gt;\u0026thinsp;0.25; quite wake by low-amplitude EEG with alpha activity [8-12Hz], low EMG and PIR\u0026thinsp;\u0026lt;\u0026thinsp;0.25. NREM sleep showed high-amplitude EEG with delta activity [1Hz-5Hz], low EMG and PIR\u0026thinsp;\u0026lt;\u0026thinsp;0.25, whereas REM sleep displayed high-amplitude EEG with theta activity [4-8Hz], flat EMG and PIR\u0026thinsp;\u0026lt;\u0026thinsp;0.25. Representative traces are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e. Time spent in each vigilance stage [min] was quantified per hour.\u003c/p\u003e\n\u003ch3\u003eEMG Signal Quantification\u003c/h3\u003e\n\u003cp\u003eEMG recordings from trapezius muscles were used to assess REM sleep without atonia (RSWA), a primary feature of RBD-like behavior. Therefore, REM sleep muscle activity was evaluated using two approaches: (1) manual scoring of REM epochs featuring elevated EMG amplitude and visible movement, and (2) automated analysis of EMG power per REM epoch using Fast Fourier Transform, normalized to EMG\u003csub\u003eNREM\u003c/sub\u003e power. REM epochs with EMG power exceeding three standard deviations above EMG\u003csub\u003eNREM\u003c/sub\u003e power were classified as showing increased REM sleep muscle activity. The manual analysis acts as a control to prevent overestimation or misinterpretation of REM sleep muscle activity caused by EMG artifacts, while the automated analysis minimizes experimenter bias inherent to manual scoring.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePower Density Analysis\u003c/h2\u003e \u003cp\u003ePower density spectra were derived from 24-hour LFP recordings using Fast Fourier Transformation with a Hanning window function by transforming the time-domain signals into a frequency-domain spectrum. This was performed through a customized analysis LabVIEW-tool (National Instruments). The power [dB] was calculated as the square of EEG magnitude [\u0026micro;V] on a logarithmic scale. With a frequency resolution of 0.5Hz, the power was plotted across the frequency range from 0.5Hz up to 100Hz.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eBehavioral Assessments\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOlfactory Buried Food Test\u003c/h3\u003e\n\u003cp\u003eFollowing an overnight fasting, the buried food-seeking test investigated the ability of mice to detect the vanilla odor originating from a cookie buried beneath 3cm clean bedding.\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Latency time [s] to retrieve the buried cookie was recorded from videos using a stopwatch. A habituation period of 2 hours allowed the mice to accommodate to a clean cage with 3cm clean bedding.\u003c/p\u003e\n\u003ch3\u003eOlfactory Discrimination Test\u003c/h3\u003e\n\u003cp\u003eOlfactory discrimination was assessed by simultaneously presenting two filter papers containing water (neutral) and vanilla aroma (Vahin\u0026eacute;, 1:100; attractive) for 2 minutes in the home cage placed inside sound-attenuated chambers.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Cumulative sniffing duration [s] directed to each stimulus was measured from video recordings using a stopwatch. Discrimination ability was defined by the difference in sniffing time [s] between water and vanilla. During testing, mice were wired to enable concurrent LFP recordings. Prior to the assay, mice were habituated to experimental conditions for 2 hours.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLight-Dark Test\u003c/h2\u003e \u003cp\u003eThe light-dark transition test, which exploits the innate aversion of mice to illuminated areas, was used to assess exploratory behavior and anxiety.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e The apparatus (40 x 40 cm) consisted of an equally sized light compartment (1000 Lux) and dark compartment connected by a small opening and equipped with infrared beam sensors for movement tracking. Mice were placed in the dark chamber and allowed to explore freely for 15 minutes. EthoVision XT Software (version 17, Noldus) automatically quantified time spent in each compartment [s] and the number of transitions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAccelerating RotaRod Motor Performance Test\u003c/h2\u003e \u003cp\u003eTo longitudinally investigate impairments in motor coordination and postural instability, mice were placed on an accelerating RotaRod apparatus (CT-ENV-575M-X4, Med Associates Inc. St. Albans). All animals underwent a training trial of five minutes before the testing protocol. The testing protocol involved three subsequent trials in which animals ran for five minutes straight on a from 4-to-40rpm accelerating rod with 15 minutes time interval. The latency [s] for each mouse to fall from the apparatus was recorded and averaged across three trials as indicator for motor coordination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCatWalk XT Gait Analysis\u003c/h2\u003e \u003cp\u003eGait abnormalities were assessed using the Catwalk XT system (Noldus). Mice freely transverse an enclosed gateway on an illuminated glass plate that captures pawprints with a high-speed video camera positioned under the gateway. Habituation to the CatWalk XT system involved allowing mice to freely transverse the gateway. The following day, mice were trained by completing three compliant runs, each with a maximum speed variation of 60%. On the experimental day, a minimum of five consecutive runs with a maximum speed variation of 25% were acquired to ensure reliable data interpretation. Catwalk XT 10.6 software enabled the calculation of an extensive range of spatial and temporal measurements.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eMouse brains were collected through anaesthetized decapitation and fixed in 10% buffered formalin for 24 hours. Samples underwent automated processing steps: 30min in formalin, 30min in water, 1h in 70% ethanol, 1h30 in 80% ethanol, 30min in 96% ethanol, 1h30 in 96% ethanol, 1h30\u0026thinsp;+\u0026thinsp;2h in 100% ethanol and 1h\u0026thinsp;+\u0026thinsp;2h in xylene. Afterwards, samples were embedded in paraffin wax at 63\u0026deg;C and cooled. Sections of 5\u0026micro;m were sliced using a Leica RM2255 microtome and dried at 50\u0026deg;C for 24 hours. Upon staining, sections were deparaffinized by sequential immersion steps: 2 x 10min xylene, 5min in 100% ethanol, 5min in 90% ethanol, 5min in 70% ethanol, 5min in 50% ethanol and 5min in water. Antigen retrieval was performed with 88% formic acid in distilled water for 20min, followed by 2.5\u0026micro;g/mL proteinase K (Roche, 03115836001) in PBS for 30min at room temperature. Endogenous peroxidase was blocked with 3% hydrogen peroxidase (DAKO, S2023) for 5min. Next, sections were incubated with primary antibodies, washed in wash buffer (DAKO, S3006) and followed by 30min incubation with secondary antibodies (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e). Target antigens were visualized during 10min incubation of DAB\u003csup\u003e+\u003c/sup\u003e chromogen (DAKO, K3468) followed by counterstaining with hematoxylin. Dehydration was conducted by immersion of 2min in 90% ethanol, 2 x 2min in 100% ethanol and 2 x 2min in xylene. Finally, slides were scanned by NanoZoomer\u0026reg; S60vs2 digital slide scanner (Hamamatsu, C16600-01) using brightfield microscopy with 40x resolution.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eImage Analysis\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eImages were acquired through HALO Link and analyzed with VisioPharm\u0026reg; Version 2024.07 x64, an AI-driven image analysis software. Automated image classification was performed using Analysis Protocol Package (APP) created from manually annotated features (eg. PK-resistant p-α-Syn neural cells, TH-positive cells) on a subset of representative samples. The APPs were trained with a U-Net deep learning model for pixel-wise image segmentation, enabling precise feature identification. After manual validation of model sensitivity and specificity, APPs were applied to all images to detect specific features. Output variables from VisioPharm\u0026reg; were further analyzed in GraphPad Prism 10.\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eBiochemical Assays\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTissue Homogenization and Bicinchoninic Acid (BCA) Analysis\u003c/h2\u003e \u003cp\u003eRight olfactory bulb, striatum, brainstem and cortex were micro-dissected from 24-month-old mice and homogenized in RIPA buffer (comprising 50 mM Tris HCl, 150 mM NaCl, 1 mM MgCl2, 1.0% NP-40 (v/v), 0.5% sodium deoxycholate (w/v), 1 mM EDTA, 0.1% SDS (w/v), pH 7.4, Sigma, R0278) supplemented with protease inhibitor (Roche, 4693159001) and phosphatase inhibitors (Roche, 4906837001). After centrifugation at 20.000g for 5min at 4\u0026deg;C, supernatant was collected and protein concentration was determined using a BCA kit (BCA Protein Assay-Kit, Thermofisher). Samples were stored at -80\u0026deg;C prior to further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSDS-PAGE Western Blotting\u003c/h2\u003e \u003cp\u003eHomogenized samples were incubated for 30min at 37\u0026deg;C with 1:50 proteinase K (Roche, 03115836001). For SDS-PAGE western blotting, 20\u0026micro;g of the PK-(un)treated samples were dissolved in NuPAGE LDS sample buffer (4X, Invitrogen\u0026trade;) supplemented with NuPAGE Sample Reducing Agent (10X, Invitrogen\u0026trade;) and boiled at 75\u0026deg;C for 5min. After separating on a NuPAGE 4\u0026ndash;12% (w/v) gradient gel (Invitrogen\u0026trade;), proteins were transferred to a nitrocellulose membrane (BioRad) using the TransBlot Turbo system. For detection of α-Syn, the membranes were treated with 4% (w/v) PFA in PBS for 30 min at room temperature. After washing in TBS-T, the membranes were blocked for 1h using 5% skim milk (w/v) in TBS-T. They were then incubated overnight at 4\u0026deg;C with primary antibodies (\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e). After washing, the membranes were exposed to horseradish peroxidase-conjugated secondary antibodies for 1h at room temperature (\u003cb\u003eSupplementary Table\u0026nbsp;1)\u003c/b\u003e. Super Signal West Dura (Thermo Fisher Scientific) was applied and chemiluminescence was imaged by Amersham\u0026trade; 600 Imager (GE Healthcare). Target protein expression was quantified by normalizing band intensities to β-actin using ImageQuantTL software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMeso Scale Discovery Two-Step-Direct Immunoassay\u003c/h2\u003e \u003cp\u003eTotal pS129-α-Syn aggregates were quantified using MJFR13-81A immunoassay on a 96-well SECTOR\u0026reg; plate (MSD, L15X1-3). Plates were coated overnight at 4\u0026deg;C with 0.5\u0026micro;g/mL MJFR13 antibody (Abcam, 168381) and washed afterwards with phosphate buffered saline (Gibco DPBS, Thermo Fisher Scientific, MA, USA) and 0.05% (v/v) Tween-20 (VWR, PA, USA). Blocking was performed with 0.1% Casein buffer (Thermo, 37528) for 2 hours at room temperature. Calibrator (recombinant pS129-α-Syn PFF) and pooled samples were thawed, diluted in RIPA buffer (Sigma, R0278) supplemented with complete protease (Roche, 4693159001) and PhosStop phosphatase inhibitors (Roche, 4906837001) and added in duplicates for overnight incubation at 4\u0026deg;C. After washing, 1\u0026micro;g/mL 81A antibody (Biolegend, 825701) was incubated for 2 hours at room temperature, followed by 1\u0026micro;g/mL of secondary Sulfo-TAG labeled secondary antibody (Meso Scale Discovery, R32AB-1) for 1 hour. After washing, 1X MSD Read Buffer T was added, and signals were measured with the MSD S600 Sector Imager. Concentrations were calculated using a 4-parameter logistic curve fit with 1/Y\u003csup\u003e2\u003c/sup\u003e weighing, excluding calibration points with relative error exceeding 20%. Final curve fit was used to interpolate raw data and determine sample concentrations [ng/mL]. Data points falling outside the linear range of the curve fit, or at or below lower limit of quantification - defined as mean blank signal plus 10 standard deviations of blank signal \u0026ndash; were excluded from analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted in GraphPad Prism version 10. To verify if Markov assumptions were met, Shapiro-Wilk test and QQ-plot were performed to check normality of the residuals. Levene\u0026rsquo;s test and pattern recognition in residuals versus predicted values plots were used to check for homo- or heteroscedasticity. Two-tailed T-tests or ANOVA tests were used when assumptions were met; otherwise, non-parametric tests were applied. Additionally, post-hoc tests were applied to explore significant differences between groups. A \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered to indicate a statistically significant difference.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eData Availability\u003c/h2\u003e \u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary material.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u0026alpha;-Syn (alpha-Synuclein), BAC (bacterial artificial chromosome), RBD (Rapid Eye Movement Sleep Behavior Disorder), PTM (post-translational modification), PD (Parkinson\u0026rsquo;s Disease)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting Interests\u003c/h2\u003e \u003cp\u003eThe authors report no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAA led the study, performed the experimental work, conducted data analyses and drafted the manuscript. AA provided daily mentorship and scientific guidance. DC and HM assisted with data analysis and scientific interpretation. WD, PV, JDPA contributed conceptual input and critically revised the manuscript. All authors approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003e We extend our sincere gratitude to Prof. Ryosuke Takahashi, Prof. Hodaka Yamakado and the members of their lab for critically reviewing this manuscript and for providing the cryopreserved heterozygous A53T-SNCA BAC embryos. We gratefully acknowledge the assistance of In Vivo Systems Research Support Staff at Johnson \u0026amp; Johnson for their contributions to animal husbandry and welfare. We want to thank Dr. Louis de Muynck for identifying the variation in mutant SNCA copy number in the A53T-SNCA BAC line, Dr. Wouter Bruinzeel for providing the amygdala extracts from a human Parkinson\u0026rsquo;s disease patient and Dr. Wim Van der Elst for statistical advice. Finally, I would like to thank Heidi Huysmans, Sofie Embrechts, Ria Biermans, Ilse Lenaerts, Ineke Fonteyn, Bart Hermans, Dr. Dina Rodrigues Martins and Marianne Borgers for their valuable support in providing excellent training.This study was supported by a Baekeland mandate [HBC.2022.0631] funded by the Flemish government agency, VLAIO [Flander Innovation \u0026amp; Entrepreneurship], the VIB-KU Leuven Center for Brain \u0026amp; Disease Research and Johnson \u0026amp; Johnson Neuroscience Department.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBraak H, Alafuzoff I, Arzberger T, Kretzschmar H, Del Tredici K. 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Mice Expressing A53T/A30P Mutant Alpha-Synuclein in Dopamine Neurons Do Not Display Behavioral Deficits. \u003cem\u003eeNeuro\u003c/em\u003e. 2024;11(2):ENEURO.0170-23.2023. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1523/ENEURO.0170-23.2023\u003c/span\u003e\u003cspan address=\"10.1523/ENEURO.0170-23.2023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Parkinson’s disease, α-Synuclein, post-translational modifications, transgenic mouse model, olfactory dysfunction, REM sleep behavior disorder","lastPublishedDoi":"10.21203/rs.3.rs-9236452/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9236452/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson’s disease is a multisystem disorder, in its prodrome characterized by a broad spectrum of non-motor symptoms, including olfactory deficits and REM sleep behavior disorder, that emerge years before the classical motor symptoms develop. Accordingly, a growing number of studies aim to generate mouse models exhibiting α-Synuclein pathology that recapitulate this prodromal phase and its progression to motor stages. This study investigated whether transgenic bacterial artificial chromosome mice carrying the human α-Synuclein gene - with the \u003cem\u003eA53T\u003c/em\u003e point mutation, two single nucleotide polymorphisms and a Rep1 polymorphism - can capture features of prodromal and late motoric Parkinson’s disease through mutant α-Synuclein overexpression.\u003c/p\u003e\n\u003cp\u003eOver a 24-month period, 20 heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e mice and 21 wild-type mice were longitudinally assessed for both non-motor and motor symptoms associated with Parkinson’s disease. EEG-EMG and local field potential recordings were performed to evaluate rapid eye movement sleep behavior disorder and stimulus-evoked neuronal activity disturbances, respectively. Additionally, we performed a behavioral phenotyping including the buried food seeking test and discriminations test for olfactory function, along with Rotarod and CatWalk assessments to evaluate motor performance. Terminal neuropathology was examined by immunohistochemistry, western blotting and a two-step direct immunoassay to correlate pathology with functional outcomes from the longitudinal study.\u003c/p\u003e\n\u003cp\u003eCharacterization of the final pathology in heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e mice revealed a \u003cem\u003eSNCA\u003c/em\u003e transgene dose-dependent overexpression of α-Synuclein monomers, exhibiting Serine129 phosphorylation and C-terminal truncation, in the olfactory bulb, striatum and cortex. However, no SDS-resistant higher-molecular-weight α-Synuclein species [≥ 198kDa] were detected unlike those observed in the Parkinson’s disease brain sample. In addition, under our testing conditions, we were unable to identify early measurable signs of olfactory dysfunction or rapid eye movement sleep behavior disorder. Moreover, they maintained their motor performance up to 24 months, and showed no substantial loss of dopaminergic neurons, compared to wild-type mice.\u003c/p\u003e\n\u003cp\u003eIn summary, our results demonstrate that overexpression of Serine129 phosphorylated and C-terminally truncated α-Synuclein monomers in heterozygous \u003cem\u003eA53T-SNCA\u003c/em\u003e mice is insufficient to drive mature fibrillar α-Synuclein aggregation, pronounced dopaminergic neurodegeneration or Parkinsonian (non-)motor symptoms. This transgenic model therefore highlights the limited ability of these post-translational modifications to initiate pathogenic processes relevant to prodromal and advanced Parkinson’s disease.\u003c/p\u003e","manuscriptTitle":"Phosphorylation and Truncation of α-Synuclein do not trigger Parkinsonian Readouts in A53T-SNCA Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-06 18:44:33","doi":"10.21203/rs.3.rs-9236452/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-26T20:32:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T22:33:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-13T13:21:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T15:03:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269627572920396125320764238309134032831","date":"2026-03-30T19:17:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104306118740563635792731030627580044838","date":"2026-03-29T10:17:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164472983080243221832189973834253857386","date":"2026-03-28T17:13:11+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-28T13:17:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-28T08:26:46+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-27T12:31:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Parkinson's Disease","date":"2026-03-26T16:15:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-parkinsons-disease","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjparkd","sideBox":"Learn more about [npj Parkinson's Disease](http://www.nature.com/npjparkd/)","snPcode":"41531","submissionUrl":"https://submission.springernature.com/new-submission/41531/3","title":"npj Parkinson's Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4e0a1f3d-48db-4d95-8403-d1bb4de4e780","owner":[],"postedDate":"April 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":65447343,"name":"Health sciences/Neurology"},{"id":65447344,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-13T20:55:44+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-06 18:44:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9236452","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9236452","identity":"rs-9236452","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}