Olfactory sensory map is perturbed in a human wild-type α-synuclein overexpressing transgenic mouse model of Parkinson’s disease

Olfactory sensory map is perturbed in a human wild-type α-synuclein overexpressing transgenic mouse model of Parkinson’s disease | 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 Olfactory sensory map is perturbed in a human wild-type α-synuclein overexpressing transgenic mouse model of Parkinson’s disease K. C. Biju, Enrique Torres Hernandez, Alison Michelle Stallings, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6890617/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2026 Read the published version in npj Parkinson's Disease → Version 1 posted 11 You are reading this latest preprint version Abstract Olfactory dysfunction, often the earliest symptom of Parkinson’s disease (PD), can precede clinical diagnosis by over 20 years, yet its mechanism and link to a-synuclein pathology remain unclear. To understand the impact of α-synuclein pathology on the topographic olfactory sensory map that supports the detection and discrimination of particular odors, we created two double transgenic mouse models (a-Syn/M72 and a-Syn/P2) expressing tagged-M72 or tagged-P2 odor receptors in a human wild-type α-synuclein over-expressing background. We demonstrated that the sensory map is disrupted in these mice. Histological analysis showed a significant reduction in M72 and P2 olfactory sensory neurons (OSNs), with altered glomerular topographies as axons converged into supernumerary glomeruli of varying size and location. These findings suggest that a-synuclein overexpression impairs the mechanism guiding the convergence of OSN axons and thus formation of a precise olfactory sensory map. As OSNs in the nasal epithelium are accessible via non-invasive biopsy, they are a potential source of prodromal PD biomarkers. Biological sciences/Neuroscience/Diseases of the nervous system/Parkinsons disease Biological sciences/Neuroscience/Diseases of the nervous system/Neurodegeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Sense of smell is often the first casualty of Parkinson’s disease (PD) (Fereshtehnejad et al., 2019 ) and its loss may be both a useful biomarker for disease onset and a clue to pathogenesis. Over 90% of PD patients exhibit olfactory dysfunction, including impairment of odor detection, identification, and/or discrimination (Wu et al., 2011 ). This prevalence is even higher than that of the cardinal sign of resting tremor (~ 75%). Furthermore, the association reported between impaired olfaction and subsequent development of PD (G. W. Ross et al., 2008 ), suggests that it is a very early sign of idiopathic PD (Haehner et al., 2007 ). In many patients, olfactory loss predates the development of clinical PD by more than 20 years (Fereshtehnejad et al., 2019 ). There is strong evidence that α-synucleinopathy, one of the hallmarks of PD, first appears in the olfactory system, followed by divergent spread of the pathology, either up to the limbic cortex or down to the lower brainstem (Adler & Beach, 2016 ; Beach et al., 2009 ). Olfactory bulb α-synucleinopathy has a high sensitivity (95%) and specificity (91%) for PD, accurately predicting the presence of α-synuclein deposits in other brain regions (Beach et al., 2009 ). Indeed, among PD’s prodromal non-motor manifestations, olfactory loss has the highest accuracy for diagnostic performance and is one of the three prodromal markers (the other two being REM sleep behavior disorder and constipation) for which the ability to predict conversion to PD is supported by the strongest evidence (Postuma & Berg, 2016 ). Moreover, recent data from the Baltimore Longitudinal Study of Aging provide new insights into a relationship between olfaction and motor function in older adults (Tian et al., 2023 ). This study revealed an association between higher olfactory function and slower decline in manual dexterity and psychomotor speed in participants aged 50 years or older (Tian et al., 2023 ). The severity of olfactory dysfunction in PD increases with disease progression (Berendse et al., 2011 ; Roos et al., 2019 ), and severe hyposmia is one of the early predictors of PD mortality (Backstrom et al., 2018 ). Yet the mechanism of this olfactory loss and the cause-and-effect relationship between olfactory dysfunction and α-synucleinopathy, as well as their linkage to dopaminergic neurodegeneration and motor symptoms in PD, are unknown. This major gap in our understanding of pathogenesis and disease progression represents a barrier to the development of strategies for more efficacious treatments. In the olfactory system, detection and discrimination of myriad odorants are achieved by approximately 1,000 discrete odor receptors, each expressed by different subsets of olfactory sensory neuron (OSNs) embedded within the olfactory neuroepithelium that lines the nose (Buck & Axel, 1991 ; Chess et al., 1994 ; Ngai et al., 1993 ; Ressler et al., 1993 ). A given OSN sends 10 to 30 receptor-bearing cilia from its dendrite into the olfactory mucus and extends a long non-myelinated axon to the ipsilateral olfactory bulb. OSNs expressing the same odor receptor project to a common glomerulus in a spatially conserved region of the bulb, synapsing with mitral and tufted cell dendrites and thereby forming a topographic olfactory sensory map that supports the detection and discrimination of particular odors (Mombaerts et al., 1996 ; Ressler et al., 1994 ; Vassar et al., 1994 ). It is not yet known whether the olfactory sensory map undergoes degeneration in PD, thereby contributing to olfactory dysfunction. Recent studies (Kim et al., 2019 ; Luk et al., 2012 ; Rey et al., 2016 ; Uemura et al., 2021 ) have clearly shown that that inoculation of α-synuclein preformed fibrils leads to cell-to-cell prion-like transmission of pathologic α-synuclein in anatomically interconnected regions, suggesting that connectivity is a determinant of susceptibility. Since axons of OSNs, the peripheral components of the stereotypic olfactory sensory map, synapse mainly with mitral cells, which harbor α-synuclein deposits in PD (Del et al., 2002 ), OSNs are highly vulnerable, making them a potential source of prodromal biomarkers. OSNs residing in the peripheral nasal epithelium may especially warrant detailed analysis, since they are developmentally related to the central nervous system, yet are accessible for diagnosis by relatively non-invasive biopsy or nasal brushing. Thus, to understand the affect of olfactory bulb α-synuclein pathology on the olfactory sensory map, we generated two double-transgenic mice that express either tagged-M72 or tagged-P2 odor receptor in a human α-synuclein over-expressing background, enabling the first demonstration of degenerative changes in the olfactory sensory map. We discuss the implications of these findings for human PD. RESULTS The olfactory sensory map Olfactory processing begins when odorant molecules bind to odorant receptors on the cilia of OSNs within the olfactory neuroepithelium lining the nasal cavity. The transduced odor signals are then relayed to the olfactory bulb. From there, the encoded information is transmitted to the olfactory cortices and subsequently to various other brain regions (Buck, 1996; Shepherd, 1994). In mice, a mature OSN typically expresses one allele (Chess et al., 1994) of one (Malnic et al., 1999) of the 1,141 discrete odorant receptor genes (Barnes et al., 2020). A given OSN sends 10 to 30 receptor-bearing cilia from its dendrite into the olfactory mucus and extends a long non-myelinated axon through the epithelium and cribriform plate to the ipsilateral olfactory bulb. OSNs expressing the same odorant receptor protein (represented by individual colors in Fig. 1) are scattered in highly overlapping and strikingly complex zones in the olfactory neuroepithelium (Zapiec & Mombaerts, 2020). However, they converge onto one or a few glomeruli in a spatially conserved region of the ipsilateral olfactory bulb (Bozza et al., 2002; Mombaerts et al., 1996; Ressler et al., 1994; Vassar et al., 1994), where they synapse with the apical dendrites of mitral cells and tufted cells, forming an olfactory sensory map that enables the detection and discrimination of specific odors. The axons of mitral cells project to a variety of more central and higher-order brain regions that mediate learned and innate odor-driven behaviors. Expression of human a -synuclein in the olfactory system of a -Syn transgenic mice Figures 2-4 combine data from a-Syn transgenic, a-Syn/M72 and a-Syn/P2 double-transgenic, and their littermate control mice. Since there is no difference either in the pattern or levels of a-synuclein among these mice, for simplicity, the mice are represented as a-Syn and Control. The pattern of expression of the transgene, human a-synuclein, in the olfactory system was assessed by immunofluorescence using antibodies specific to human a-synuclein protein. No human a-synuclein immunofluorescence was detected in the olfactory epithelium that lines any of the endoturbinates or ectoturbinates in the nasal cavity of either a-Syn or Control mice. Representative images of endoturbinate I are shown in Fig. 2. In the olfactory bulb of a-Syn mice, intense human a-synuclein immunofluorescence was detected in all layers except the superficial olfactory nerve layer that is comprised primarily of axons of the olfactory sensory neurons (Fig. 3). The immunofluorescence was localized primarily to the neuronal processes. However, the soma of some mitral cells showed intense human a-synuclein immunofluorescence. As expected, no immunofluorescence to human a-synuclein was observed in age-matched Controls (Fig. 3H). We also confirmed the presence of human a-synuclein in the olfactory bulbs of a-Syn mice using immunoblotting. A single band at ~15 kDa, corresponding to monomeric human a-synuclein, was detected in a-Syn mice, whereas no human a-synuclein was detected in Control mice (Fig. 4A). Since the majority of pathological a-synuclein is phosphorylated at serine 129 (pSer129), we also performed immunoblotting using antibodies to pSer129-a-synuclein. As was the case for monomeric human a-synuclein, pSer129-a-synuclein was detected only in a-Syn mice (Fig. 4A). Since the antibody specific to human a-synuclein detects only monomeric a-synuclein, we also used a second antibody that binds to both human and mouse a-synuclein and detects all forms of a-synuclein. Use of this antibody revealed, in addition to monomeric a-synuclein at ~15 kDa, a variety of forms of a-synuclein — ranging from 30kDa to over 250 kDa — in a-Syn mice. However, in the Control mice only monomeric a-synuclein was detected, albeit unsurprisingly to a lesser level (Fig. 4E). In PD, a-synuclein undergoes conformational changes to form toxic insoluble aggregates, a neuropathological feature that is associated with disease progression. To assess the accumulation of abnormal a-synuclein aggregates in the olfactory bulb, TBS-soluble and TBS-insoluble (soluble in SDS and urea) fractions of a-synuclein were collected through sequential extraction with high-speed ultracentrifugation (Bandopadhyay, 2016) followed by immunoblotting using the antibody that binds to both human and mouse a-synuclein. As expected, monomeric a-synuclein was present in the TBS-soluble fraction from both a-Syn and in a smaller amount from Control mice (Fig. 4F). However, the other higher molecular weight forms — ranging from 30kDa to 250 kDa — were present only in the TBS-soluble fraction from a-Syn mice. Similarly, various forms of a-synuclein in the SDS- and urea-soluble fractions were detected only in a-Syn mice (Fig. 4F). It has been suggested that the SDS solubilizes a-synuclein oligomers that can include membrane bound forms, whereas urea denatures the insoluble aggregated and fibrillar or amyloidogenic forms of a-synuclein (Anderson et al., 2006; Bandopadhyay, 2016). Lastly, we assessed expression levels of both human and mouse a-synuclein mRNA in the olfactory bulb using RT-qPCR. Whereas human a-synuclein mRNA was detected only in a-Syn mice, mouse a-synuclein mRNA was detected, in similar levels, in both a-Syn and Control mice (Fig. 4G), indicating that the overexpression of human a-synuclein mRNA had no impact on the levels of endogenous mouse a-synuclein mRNA in a-Syn mice. Alteration of olfactory sensory map in a -Syn/M72 and a -Syn/P2 double transgenic mice To examine the effect of a-synuclein overexpression on the olfactory sensory map, 12-month-old a-Syn/M72 and P2 and littermate Control/M72 and P2 mice were processed for whole-mount X-Gal staining (n=16/genotype). In the Control/M72 mice, the M72 olfactory sensory neurons (OSNs) were readily visualized by blue staining, and their axons converge on two glomeruli, one on the lateral and one on the medial aspect of each olfactory bulb (Fig. 5A). a-Syn/M72 mice exhibited a drastic reduction in the number of M72 OSNs. Consequently, OSN axonal projections to the bulb were dramatically reduced (Fig. 5B-E), leading to formation of small glomeruli (Fig. 6C). While the OSN axonal projection patterns of all a-Syn/M72 showed changes in both olfactory bulbs (Fig. 5B, C, D), many mice showed disproportionate unilateral changes (Fig. 5C), which is highly intriguing since cardinal motor symptoms of PD are usually unilateral at the earliest stage. As seen for the a-Syn/M72 mice, P2 OSNs and their axons were dramatically reduced in the a-Syn/P2 mice (Fig. 5J-M). Consequently, the P2 glomeruli in a-Syn/P2 mice were smaller and often their staining intensity was lighter, compared to Control/P2 mice (Fig. 6B, D). Although the M72 and P2 OSNs in a-Syn/M72 and P2 mice project broadly to similar locations in the bulb as their counterparts in Control mice, unlike the tight glomerular organization in Controls (two glomeruli per bulb — one lateral and one medial), supernumerary glomeruli of varying size were common in the a-Syn/M72 and a-Syn/P2 mice (Fig. 7 and Table 1). The positions of individual glomeruli (e.g. M72 or P2 glomeruli) have been shown to be topographically defined. However, there was no consistency between individual mice in the location of these supernumerary glomeruli (Fig. 7), indicating that a-synuclein overexpression affects the mechanism guiding the convergence of axons of OSNs expressing a particular odorant receptor. Genotype (n=10/genotype) Glomeruli/bulb (Min—Max) M72 2.1 (2—3) a-Syn/M72 4.3 (3—6) P2 2 (2—2) a-Syn/M72 3.4 (3—4) Table. 1: Number of odorant receptor-specific glomeruli per olfactory bulb. n=20 bulbs from 10 mice per genotype. Olfactory dysfunction in a -Syn/M72 and a -Syn/P2 double transgenic mice The human wild-type a-synuclein overexpressing transgenic mice have previously been shown to exhibit olfactory dysfunction (Biju et al., 2025; Fleming et al., 2008). To confirm olfactory dysfunction in the double transgenic mice, a-Syn/M72 and a-Syn/P2 (together represented as a-Syn mice in Fig. 8), and their respective littermate controls (n=5/genotype) were subjected to a battery of olfactory behavioral tests. The a-Syn mice took significantly more time than Control mice to retrieve hidden cereal (Fig. 8A), a measure of hyposmia. The time to find an exposed piece of cereal was not significantly different between a-Syn and Control mice (Fig. 8B). In test for the ability to discriminate between two odors using a habituation/dishabituation paradigm, the a-Syn mice exhibited deficits in odor discrimination (Fig. 8C), seen as decreased exploratory time, compared with Control mice, upon presentation of a second odor following habituation to a first odor. We also used a paradigm based on the time spent in familiar versus unfamiliar compartments to assess olfactory dysfunction. This task relies on rodents typically preferring locations marked with their own scent (familiar compartment). Consequently, mice with normal olfaction will spend more time in a familiar compartment when choosing between familiar and unfamiliar compartments. The test further confirmed olfactory dysfunction in a-Syn mice, as indicated by the equal amounts of time spent in familiar and non-familiar compartments (lack of preference for familiar compartment; Fig. 8D). Since the number of M72 and P2 OSNs was dramatically lower in the odorant receptor tagged double transgenic mice, we also assessed olfactory sensitivity. While the lowest dilution of the odorant at which the exploratory time of Control mice was significantly different compared with mineral oil (diluent) was 10 -4 , the lowest dilution for a-Syn mice was 10 -2 , indicating a drastically reduced olfactory sensitivity in a-Syn mice (Fig. 8E). Since olfactory behavioral tests are influenced by the overall activity levels of the animals, and since a-Syn mice exhibit hyperactivity at younger ages, we assessed the general activity levels (speed and distance travelled) of the mice in their home cages. Although there was a temporal difference in activity levels between a-Syn and Control mice during the dark cycle (Fig 8F, I), total activity levels were similar during both the dark (Fig 8G, J) and light (Fig. 8H, K) cycles (when the olfactory tests were performed). DISCUSSION We generated two separate odorant receptor-tagged mice in a human α-synuclein over-expressing background (Line 61) and showed that there was a dramatic decrease in the number of OSNs and perturbations in the stereotypic convergence of their axons in the olfactory bulbs, suggesting that neurodegenerative changes in the olfactory sensory map contribute to olfactory dysfunction in PD. Except for a recent study that computed the total number of OSNs in an aged human specimen (n of 1) to be ~ 2.7 million (Low et al., 2024), to our knowledge, no data exist to assess whether there is a reduction in the number of OSNs in PD patients. This lack of data is unsurprising given the technical challenges associated with assessing the total number of OSNs in humans, especially given that the olfactory epithelium in humans is patchy and interspersed with respiratory epithelium (Holbrook et al., 2011 ). However, our extrapolation, based on the number of OSN in mouse olfactory epithelium and on glomerular data in human PD patients, predicts that the number of OSNs in PD patients is likely to be dramatically lower than that of healthy subjects. For example, analysis of human olfactory bulbs revealed that global glomerular voxel volume of olfactory bulbs in PD patients is half that of control patients (Zapiec et al., 2017 ). This study further showed that the higher the α-synuclein load in the olfactory bulb, the lower the global glomerular voxel volume, suggesting a causal relationship between α-synuclein pathology and glomerular voxel volume. In studies of odorant receptor-tagged mice, researchers have demonstrated a strong linear correlation between OSN number and glomerular voxel volume (Bressel et al., 2016 ). Based on this, it has been suggested that glomerular voxel volume can be used as a surrogate marker for the number of OSNs (Bressel et al., 2016 ). Together, these reports suggest that the significant reduction in global glomerular voxel volume in the olfactory bulb of PD patients corresponds to a significant reduction in the number of OSNs in the nasal epithelium, as seen in α-Syn/M72 and α-Syn/P2 mice in the current study. Thus, OSNs from PD patients warrant in-depth analysis for transcriptomic and metabolomic signatures, as it could provide crucial insight into the pathogenesis of PD. An important question is to what extent are our data on M72 and P2 OSNs relevant to OSNs expressing other odorant receptors in the nasal epithelium of PD patients. There are between 4 to 8 million total OSNs in the olfactory epithelium of an adult mouse, and each of their olfactory bulbs contains about 3,600 to 3900 glomeruli (Richard et al., 2010 ), making it technically challenging to assess subtle neuroanatomical changes. However, distributed in two non-overlapping zones (Zolfr160 and Zolfr17) in the olfactory epithelium (Zapiec & Mombaerts, 2020 ), the absolute number of M72- and P2-expressing OSNs in a young adult mouse is only about 6,000 and 12,000, respectively, and typically, they each project to only two glomeruli in each bulb (Biju et al., 2008 ; Bressel et al., 2016 ). Because only a small proportion of the total OSNs express either M72 or P2 odorant receptors, and they project to only two glomeruli each, it may even be possible to reveal subtle neuroanatomical olfactory system changes, thereby facilitating precise definition of the structural changes in the olfactory epithelium and olfactory bulb that lead to impaired sense of smell in Parkinson’s disease. This theory is based on the hypothesis that olfactory dysfunction in PD affects all odors, and thus a global change in OSN number and projection pattern will be faithfully reflected by the M72 and P2 OSNs. Indeed, Doty et al. first observed that PD features a general olfactory deficit, rather than a deficit to certain specific odors (Doty et al., 1988 ). Subsequently, there were reports showing the opposite — that PD patients experience reduced sensitivity to certain specific odors such as banana, pineapple, pizza, licorice, dill pickle, cinnamon, wintergreen, smoke, and gasoline (Bohnen et al., 2007 ; Double et al., 2003 ; Hawkes & Shephard, 1993 ). However, more recent studies (Chou & Bohnen, 2009 ; Doty, 2012 ; Hahner et al., 2013 ; Morley et al., 2018 ; Vaswani et al., 2023 ) have confirmed the original findings of Doty et al. (Doty et al., 1988 ) that olfactory dysfunction in PD is not restricted to specific odors. In fact, a recent study that used eight machine learning models to distinguish PD hyposmia from non-PD hyposmia showed that PD-related hyposmia does not exhibit a unique pattern of odor selectivity distinct from general hyposmia (Mitchell et al., 2025 ). Together, these studies clearly establish that olfactory dysfunction is unrelated to specific odorants; however, it is possible that the extent of damage may vary among OSNs expressing a particular odorant receptor. For example, we found that 76% M72 OSNs were lost in the α-Syn/M72, whereas only 45% of P2 OSNs were lost in the α-Syn/P2. The exact reason for this differential loss is unclear. One possibility is that axons of M72 OSNs need to travel longer distances to reach their glomeruli in the bulb, making these unmyelinated axons more vulnerable to α-synuclein induced toxicity. Sense of smell is overwhelmingly the first casualty of PD, often predating the development of clinical PD by over 20 years (Fereshtehnejad et al., 2019 ); and since olfactory dysfunction is likely an early victim of the same pathological mechanism that eventually leads to PD’s motor symptoms, it may be a window into pathogenesis. However, a major roadblock to understanding olfactory dysfunction in human prodromal PD patients is the very fact that there is currently no way to identify prodromal patients for such studies. Moreover, the molecular pathology in post-mortem human brain samples may no longer reflect what occurred in the prodromal stage of PD. However, the mechanism of PD-related olfactory deficits can be effectively explored through the use of mouse models, since the neural mechanisms of odor processing in rodents are well understood and share important characteristics with humans. To best represent prodromal PD, we used 12-month-old mice for all studies, for these reasons: (1) Age is the greatest risk factor for PD, and it is conceivable that young mice are capable of mounting a sufficient defense against toxic insults (Borghammer & Van Den Berge, 2019 ). (2) According to The Jackson Laboratory, 12 months in mice is the equivalent of 45 years in humans; since the average age of clinical diagnosis of PD is 65, and olfactory loss can occur over 20 years prior to clinical diagnosis, 45 can be considered reasonably within the prodromal period. (3) Twelve months of age in α-Syn mice is considered a prodromal stage (Richter et al., 2023 ). While there are reports of α-Syn mice exhibiting “motor deficits” at earlier ages (Rabl et al., 2017 ), these “motor deficits” are actually deficits in motor learning, motor coordination or both and should not be confused with the typical motor symptoms of clinical PD. The term “motor dysfunction” includes: (a) deficits in motor learning, (b) deficits in fine motor skills/motor coordination, and (c) gross deficits in ambulation/gait. Depletion of striatal dopamine alone is sufficient to induce deficits in motor learning and fine motor skills/motor coordination; consequently, these deficits (e.g., in handwriting, dexterity, etc.) are evident in the prodromal stage. However, the gross motor deficit in ambulation/gait that patients exhibit at the time of clinical diagnosis require depletion of both striatal and nigral dopamine (Gonzalez-Rodriguez et al., 2021 ). Similarly, α-Syn mice exhibit striatal dopamine loss and deficit in motor learning/fine motor tasks during motor coordination tests; however, nigral dopaminergic neuronal loss and gait dysfunctions are not observed in α-Syn mice at 12 months of age, which is why they are considered a prodromal model of PD. The exact mechanism for the observed changes in the olfactory sensory map in our double-transgenic mice is unclear. OSNs continuously regenerate, and the newly formed neurons project their axons to the olfactory bulb throughout the lifespan of the animal (Brann & Firestein, 2014 ). Thus, connections to the olfactory bulb must be reestablished continuously to maintain a precise olfactory sensory map. Our data show a dramatic reduction in the number of OSN and supernumerary glomeruli, indicating that both regeneration of OSNs and targeting of their axons are affected. It has been previously shown that both the peripheral development and target destination of OSNs are influenced by the activity of mitral cells in the olfactory bulb (Biju et al., 2008 ). In mice having a gene-targeted deletion of the Shaker potassium ion channel (Kv1.3), mitral cells have altered action potential and increased firing frequency (Fadool et al., 2004 ). M72 and P2 mice in Kv1.3-null background also exhibit a drastic reduction in OSN numbers, as well as supernumerary glomeruli (Biju et al., 2008 ). Indeed, mitral cells overexpress human α-synuclein in our α-Syn/M72 or P2 double transgenic mice. α-Synuclein is found in axon terminals and plays an active role in the downregulation of neurotransmitter release (Nemani et al., 2010 ). Thus, overexpression of human α-synuclein could disrupt mitral cell activity, causing perturbations in the olfactory sensory map as seen in the Kv1.3-null. Of note, over-expression of normal α-synuclein (vs. mutant form) is sufficient to cause PD, and a gene-dosage effect exists in that triplication results in higher α-synuclein levels, more severe pathology, and earlier onset than duplication (Fuchs et al., 2007 ; O. A. Ross et al., 2008 ; Singleton et al., 2003 ). Although we focused on the olfactory system, α-Syn mice harbor α-synuclein pathology in many other brain areas (Rockenstein et al., 2002 ) as seen in PD. Therefore, we cannot completely exclude potential contributions of extra-olfactory bulb α-synuclein pathology on the observed effect in the olfactory sensory map. In summary, we provide compelling evidence for perturbations in the olfactory sensory map of a human wild-type a-synuclein overexpressing transgenic mouse model of PD, and for their relevance to understanding olfactory dysfunction in PD. If olfactory dysfunction in PD is accompanied by (whether or not due to) degenerative changes in the topographic olfactory sensory map, these early structural/cellular changes should be accompanied by the appearance of disease-specific changes in the molecular atlas of the OSNs in the nasal epithelium. Mapping of this molecular atlas would not only yield minimally invasive biomarkers for prodromal prognostication of the disease, but may also lead to insights into both disease pathogenesis and disease progression, which in turn could help development of new neuroprotective strategies. METHODS Mice Three different kinds of transgenic mice in C57BL/6J background were used: (1) mThy1-hSNCA (Line 61), (2) M72-IRES-tau-LacZ, and (3) P2-IRES-tau-LacZ. The mThy1-hSNCA mice (henceforth, α-Syn mice) express the human wild-type α-synuclein gene under the direction of the mouse thymus cell antigen 1 (thy1) promoter (Rockenstein et al., 2002 ). These mice are useful for studying PD pathogenesis and neurodegeneration elicited by the toxic effects of aggregation and somatic accumulation of human α-synuclein. The mThy1-hSNCA mice were a generous gift from Dr. Randy Strong (University of Texas Health Science Center at San Antonio, TX). M72-IRES-tau-LacZ and P2-IRES-tau-LacZ mice (henceforth, M72 and P2 mice) were generated previously (Mombaerts et al., 1996 ; Zheng et al., 2000 ) via placement of an internal ribosome entry site (IRES) directing the translation of tau-lacZ fusion protein immediately downstream of the M72 (Olfr160) or P2 (Olfr17) odorant receptor stop codon. In these genetically altered strains of mice, olfactory sensory neurons that transcribe the modified M72 or P2 receptor allele also express tau-lacZ in their cell bodies and axons, enabling direct visualization of individual olfactory sensory neurons in the epithelium, as well as the pattern of projections of these neurons in the olfactory bulb, by histological staining for β-galactosidase (X-Gal) activity. The M72 and P2 mice were a generous gift from Dr. Debra Ann Fadool (Florida State University, FL). We generated double-transgenic M72 and P2 odorant receptor-tagged mice that over-express human wild-type α-synuclein (α-Syn/M72 or α-Syn/P2 mice) by breeding homozygous M72 and P2 mice with hemizygous α-Syn mice. All the double-transgenic mice used in this study were hemizygous for human wild-type α-synuclein and homozygous for the modified M72 or P2 receptor allele. Since the transgene (human wild-type α-synuclein) in α-Syn mice is inserted into the X chromosome, female mice exhibit diminished and variable PD deficits (Biju et al., 2025 ; Chesselet et al., 2012 ; Grant et al., 2014 ), likely due to random inactivation of the transgene-bearing chromosome in hemizygous females during early embryogenesis. Therefore, we used male α-Syn/M72 and α-Syn/P2 mice, at 12 months of age, for all our studies. The mice were housed in standard plexiglass cages with 7099 TEK-Fresh animal bedding (Envigo, Indianapolis, IN) and were fed a standard Teklad irradiated LM-485 mouse diet (Cat # 7912; Envigo, Indianapolis, IN) ad libitum and with full-time access to acidified drinking water. The mice were maintained in a 12/12 hour light/dark cycle at 24 O C room temperature and 50–55% humidity. Animal husbandry was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Society for Neuroscience Policies on the Use of Animals and Humans in Research. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the South Texas Veterans Health Care System, Audie L. Murphy Division, and the University of Texas Health Science Center, San Antonio. The smallest possible number of mice was used (based on power calculations) and all efforts were made to minimize suffering. All experiments were randomized and performed blind-coded whenever feasible. Olfactory behavioral tests The olfactory behavioral tests were performed in a dedicated rodent behavior-testing room (sound attenuated and with HEPA-filtered air) during the light cycle with 30–40 lux light intensity and 24 0 C room temperature maintained throughout the tests. General anosmia was assessed as described before (Biju et al., 2020 ; Biju et al., 2025 ) by recording latency to retrieval of a cereal hidden under the bedding. Olfactory discrimination was assessed by either recording of exploratory time upon presentation of a second odor (amyl acetate — banana flavor) following habituation to a first odor (peppermint) or by recording the time spent in familiar vs. non-familiar compartments using our published protocol (Biju et al., 2018 ). Olfactory sensitivity was assessed by recording exploratory time to one of five dilutions of odorant (10 − 1 , 10 − 2 , 10 − 3 , 10 − 4 , and 10 − 5 dilutions of amyl acetate in mineral oil) for one 3-minute session using a published method (Witt et al., 2009 ). Briefly, the mice were acclimated individually to a clean standard mouse cage (without bedding) with a piece of filter paper enclosed in a tissue cassette for four sessions of 15 minutes each. Immediately after the acclimation the mice were introduced to the test cage with a tissue cassette containing a filter paper dampened with equal volumes of either mineral oil or one of the five dilutions of amyl acetate, and the sniffing time was recorded in a three-minute session. After completing the initial three-minute session, for all mice were immediately started on the second three-minute session with the next dilution of odorant, chosen randomly, and so on until all the five dilutions of amyl acetate and mineral oil were completed. Home cage activity monitoring General activity in the home cage was recorded using the ActiMot2 infrared light beam activity monitoring system (25 beams each on X and Z directions and 16 beams on Y direction; TSE Systems, Inc., Chesterfield, MO) using our published protocol (Biju et al., 2020 ). The home activity of the mice was recorded at a sampling rate of 100 Hz with a recording interval of ten minutes for 24 hours continuously. Speed and total distance travelled during the light and dark phases were analyzed and compared among the groups. Immunofluorescence The mice were anesthetized with an overdose of ketamine HCl/xylazine HCl solution and perfused transcardially with 10–20 ml ice-cold phosphate-buffered saline (PBS, pH 7.4) followed by an equal volume of ice-cold 4% paraformaldehyde in PBS. Brains were removed and post-fixed overnight in the same fixative at 4 0 C. Parts of the heads containing nasal epithelium were first post-fixed overnight in the same fixative and then decalcified in 0.3 M EDTA for 48 hours at 4 0 C. The tissues were cryoprotected in sequential solutions of sucrose (10% for 2 h, 20% for 2 h, and 30% for 24–48 h), and then embedded in Tissue-Tek OCT compound. Cryosections of olfactory epithelia and bulbs were prepared at 15 µm thickness in the coronal plane using a Leica CM 1950 cryostat. Immunofluorescence was performed as described (Biju et al., 2010 ). Briefly, the sections were incubated with an antibody (1:2,000 dilution) specific for human a-synuclein (cat # ab138501; abcam, Waltham, MA) overnight at 4 0 C. The secondary antibodies were conjugated with Alexa Fluor® 488 (1:200; Molecular Probes). Sections were coverslipped with VECTASHIELD Vibrance® Antifade Mounting Medium with DAPI (cat # H-1800; Vector Laboratories, Inc., Newark, CA). Fluorescent images were analyzed with a Keyence BZ-X800 microscope. Stringent control procedures were utilized to ensure specificity of immunofluorescence. X-Gal staining The mice were anesthetized and then perfused transcardially as described above for immunofluorescence. The heads were removed, deskinned and post-fixed overnight in the fixative at 4 0 C. The reagents and protocol for X-Gal staining have been described (Biju et al., 2008 ; Mombaerts et al., 1996 ). Briefly, the heads were decalcified in 0.3 M EDTA for 3 days at 4 0 C and then the nasal epithelia and olfactory bulbs were exposed by removing the skull from the dorsal side. The heads were washed with Buffer A for 5 minutes and then again for 25 minutes. The heads were then incubated twice with Buffer B for 5 minutes each, followed by an 8-hour incubation in Buffer C. Following a 10-minute fixation in 4% paraformaldehyde and then a wash in PBS, whole mount images were captured using a stereomicroscope. Thereafter, parts of the heads containing nasal epithelium and olfactory bulbs were cryoprotected in 30% sucrose, frozen in OCT and then cryosectioned at 30 µm thickness. The cryosections were counterstained with neutral red and used for manual counting of M72 and P2 olfactory sensory neurons (Biju et al., 2008 ). Immunoblotting Immunoblot analysis of a-synuclein in the olfactory bulb was performed using our published protocol (Biju et al., 2025 ). Briefly, following euthanasia by CO 2 inhalation, the mice were decapitated. The olfactory bulbs were quickly dissected out, snap frozen in liquid nitrogen, and then processed for protein extraction. Protein concentrations were determined through Bradford assay. Samples (20 mg) were loaded on Invitrogen Novex 12% Tris-Glycine Plus Midi Gels (cat # WXP01226BOX; ThermoFisher Scientific, Waltham, MA), electrophoresed for 50 minutes at 150 volts, and then transferred onto nitrocellulose membranes. The membranes were stained with Ponceau S to confirm equal loading. The membranes were blocked with 5% nonfat dried milk in 1X TBST buffer and then incubated overnight with one of the following antibodies, prepared in 5% nonfat dried milk, at 4 0 C: (1) human a-synuclein (cat # ab138501; abcam, Waltham, MA), (2) human a-synuclein phosphorylated at serine 129 (cat # ab168381; abcam), (3) human and mouse a-synuclein (cat # ab212184; abcam), (4) GAPDH (cat # G9545; Sigma-Aldrich, Inc. St. Louis, MO), or (5) b-actin (JLA20; cat # AB_528068; DSHB, University of Iowa). The bands were visualized using Pierce ECL Western Blotting Substrate (Cat # 32209; ThermoFisher Scientific). Optical density measurements of the bands were performed using NIH ImageJ software. Analysis of levels of soluble and insoluble a-synuclein in the olfactory bulb was performed by sequential extraction of proteins in Tris-buffered saline (TBS), sodium dodecyl sulphate (SDS) buffer, and urea buffer using a published method (Bandopadhyay, 2016 ). Subsequent immunoblot analysis of the TBS, SDS, and urea fractions of the proteins was performed as described above; however, the protein bands were visualized using SuperSignal West Pico Plus Chemiluminescent Substrate (cat # 34580; ThermoFisher Scientific). Reverse transcription-quantitative real-time PCR (RT-qPCR) analysis RNA from snap-frozen olfactory bulbs was isolated using TRIzol/chloroform extraction and RNeasy Mini Kit (cat# 74106; QIAGEN Science, Germantown, MD) (Biju et al., 2025 ). RNA was converted into cDNA using the SuperScript VILO cDNA Synthesis Kit (cat# 11754250; Invitrogen/ThermoFisher Scientific) and qPCR was performed using pre-designed TaqMan probes (ThermoFisher Scientific) for human a-synuclein (Hs00240906_m1), mouse a-synuclein (Mm01188700_m1) and b-actin (Mm02619580_g1) on QuantStudio 3 (ThermoFisher Scientific). Transcriptional levels were determined using ∆∆Ct method, and mRNA percentage relative to b-actin in Control mice was calculated. Statistics Statistical analyses of the data were performed in R version 3+ (Vienna, Austria) or GraphPad Prism 10 (GraphPad Software, Inc.). As specified in the legend for each figure, either independent unpaired t-tests or two-way repeated measures ANOVA were used. All F values and degrees of freedom used for ANOVA are also specified in the legends of respective figures. All sets of continuous data were tested for normality using the Shapiro-Wilk test. Results were expressed as mean ± SEM, and differences in mean were considered significant at P < 0.05. DATA AVAILABILITY All raw data and any additional information on methodology or data analyses used in the current study will be available upon request to the corresponding authors. Declarations COMPETING INTERESTS All authors declare that they have no financial or non-financial competing interests. Author Contribution RAC and KCB conceived and designed the study. KCB, ETH, ACF-O, and AMS performed the behavioral tests, X-Gal staining, histology, quantification of OSNs, and RT-qPCR. SKH, AMS, and ETH analyzed the immunoblot and behavioral data. ETH, KCB, and SKH designed and performed the statistical analysis. All authors were involved in overall data analysis. KCB and RAC wrote the manuscript. All authors read, commented, and approved the final manuscript. ACKNOWLEDGEMENTS This work was supported by a Merit Review Grant from the Veterans Health Administration (1 I01 BX 003157) and a grant from the William and Ella Owens Medical Research Foundation, both awarded to RAC, as well as pilot and career development grants from the Perry & Ruby Stevens Parkinson’s Disease Center of Excellence, awarded to KCB. We thank Dr. Debra Ann Fadool at Florida State University and Dr. Randy Strong at UTHSCSA for providing the original breeding pairs of M72/P2 odorant receptor tagged and a-synuclein transgenic mice, respectively. References Adler, C. H., & Beach, T. G. (2016). Neuropathological basis of nonmotor manifestations of Parkinson's disease. Mov Disord , 31 (8), 1114–1119. https://doi.org/10.1002/mds.26605 Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J., Kling, K., Lee, M., Diep, L., Keim, P. 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Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit. Neuron , 26 (1), 81–91. http://www.ncbi.nlm.nih.gov/pubmed/10798394 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2026 Read the published version in npj Parkinson's Disease → Version 1 posted Editorial decision: Revision requested 13 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers agreed at journal 11 Aug, 2025 Reviews received at journal 11 Aug, 2025 Reviewers agreed at journal 04 Aug, 2025 Reviews received at journal 31 Jul, 2025 Reviewers agreed at journal 07 Jul, 2025 Reviewers invited by journal 06 Jul, 2025 Editor assigned by journal 16 Jun, 2025 Submission checks completed at journal 16 Jun, 2025 First submitted to journal 13 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6890617","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":486548701,"identity":"f25baf0e-0dc4-4041-a345-ce8e02cfc7a7","order_by":0,"name":"K. C. Biju","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAw0lEQVRIiWNgGAWjYDACCSDmYbCBMMDgAHFa0hh4SNVymAQt/LObnz1423Y+cb90+8OPP2oY5PhuJBCw5M4xc8O5bbcTe2TOGEvzHGMwliSkxUAiwUyaF6RFIoeNmYGNIXEDYS3p34BazgG1pD9j/PGPoZ4ILTkgWw4AtSSYMfC2MSQYEPTLjZwyyTnnko17buQYS/P2SRjOPPMAvxb+GenbJN6U2cm2z0gHhtg3G3m+4wRswbCVNOWjYBSMglEwCrADAIGjQrTsIyrjAAAAAElFTkSuQmCC","orcid":"","institution":"The University of Texas Health Science Center at San Antonio","correspondingAuthor":true,"prefix":"","firstName":"K.","middleName":"C.","lastName":"Biju","suffix":""},{"id":486548702,"identity":"78acab8a-f5fe-48fc-8706-5ff5e38632d0","order_by":1,"name":"Enrique Torres Hernandez","email":"","orcid":"","institution":"The University of Texas Health Science Center at San Antonio","correspondingAuthor":false,"prefix":"","firstName":"Enrique","middleName":"Torres","lastName":"Hernandez","suffix":""},{"id":486548703,"identity":"a7d5b307-028c-4772-a419-ea4837db6120","order_by":2,"name":"Alison Michelle Stallings","email":"","orcid":"","institution":"The University of Texas Health Science Center at San Antonio","correspondingAuthor":false,"prefix":"","firstName":"Alison","middleName":"Michelle","lastName":"Stallings","suffix":""},{"id":486548704,"identity":"6c338226-bac3-4201-a9c0-27867488dd6d","order_by":3,"name":"Ada C. Felix-Ortiz","email":"","orcid":"","institution":"The University of Texas Health Science Center at San Antonio","correspondingAuthor":false,"prefix":"","firstName":"Ada","middleName":"C.","lastName":"Felix-Ortiz","suffix":""},{"id":486548707,"identity":"f784fca0-78e8-4ed8-9f41-fa3cbc7d093a","order_by":4,"name":"Skanda K. Hebbale","email":"","orcid":"","institution":"The University of Texas Health Science Center at San Antonio","correspondingAuthor":false,"prefix":"","firstName":"Skanda","middleName":"K.","lastName":"Hebbale","suffix":""},{"id":486548709,"identity":"55eb4071-5f62-4238-82a7-02e20daf9a66","order_by":5,"name":"Robert A. Clark","email":"","orcid":"","institution":"South Texas Veterans Health Care System","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"A.","lastName":"Clark","suffix":""}],"badges":[],"createdAt":"2025-06-13 20:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6890617/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6890617/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41531-026-01288-w","type":"published","date":"2026-02-10T15:57:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86919371,"identity":"f49886a9-0971-42ab-b95d-376866ca1f1f","added_by":"auto","created_at":"2025-07-17 07:22:09","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":385392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the olfactory system. A\u003c/strong\u003e Head of a P2 mouse, sectioned along the midline, showing the nostril (NS), olfactory epithelium (OE), olfactory bulb (OB), and the rest of the brain. \u003cstrong\u003eB\u003c/strong\u003e Simplified schematics of the olfactory sensory map.\u003cstrong\u003e \u003c/strong\u003eOlfactory epithelium and the major layers of the olfactory bulb are shown. Olfactory sensory neurons in the OE that express the same odorant receptor (represented by individual colors) converge to synapse onto a specific glomerulus in the olfactory bulb. CP, cribriform plate; EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/ebc0b3541bf511e9912e629c.jpeg"},{"id":86920409,"identity":"49fb7133-5f00-4018-b9e1-a57d4393ff9e","added_by":"auto","created_at":"2025-07-17 07:30:09","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":292519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLack of human a-synuclein protein expression in the olfactory epithelium (OE).\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Low magnification image of transverse section of the head, showing nasal cavities (NC), nasal septum (NS), and olfactory epithelia on endoturbinates I and II. The rectangle represents the area of endoturbinate I from which the higher magnification images of the olfactory epithelia in \u003cstrong\u003eB-G\u003c/strong\u003ewere taken. The outlines of olfactory epithelia in \u003cstrong\u003eB\u003c/strong\u003e are marked with dotted white lines. Representative images of n=4/genotype. SO, septal organ.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/54839f972e4b191482d5ba70.jpeg"},{"id":86919372,"identity":"69988cc4-cad4-4b2d-b1e4-1c270096c7f7","added_by":"auto","created_at":"2025-07-17 07:22:09","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":757333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePattern of human a-synuclein protein expression in the olfactory bulb.\u003c/strong\u003e \u003cstrong\u003eA-C\u003c/strong\u003e Low-magnification image of transverse section of the olfactory bulb from a-Syn mice, showing DAPI-stained nuclei and human a-synuclein immunofluorescence. \u003cstrong\u003eD-F\u003c/strong\u003e Higher magnification images of the olfactory bulb from a-Syn mice showing a single mitral cell with human a-synuclein immunofluorescence in the soma and in the dendritic processes (arrowhead). \u003cstrong\u003eG-I\u003c/strong\u003eLow-magnification image of the olfactory bulb from Control mice, showing absence of human a-synuclein immunofluorescence. Representative images of n=4/genotype. EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MCL, mitral cell layer; ONL, olfactory nerve layer.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/b6bbcd7fc1d9c4c165521806.jpeg"},{"id":86919383,"identity":"1431877f-2d39-4cdf-964d-01be73fe6fd8","added_by":"auto","created_at":"2025-07-17 07:22:09","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":324400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunoblot and RT-qPCR analyses of human a-synuclein transgene and endogenous mouse a-synuclein in the olfactory bulb.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Immunoblot showing monomeric a-synuclein and pSer129-a-synuclein exclusively in a-Syn mice, using antibodies specific to human a-synuclein. \u003cstrong\u003eB-D\u003c/strong\u003e Histogram plots of the optical density measurement of a-synuclein and GAPDH in \u003cstrong\u003eA\u003c/strong\u003e. \u003cstrong\u003eE\u003c/strong\u003e Immunoblot showing various forms of a-synuclein, using an antibody that detects both human and mouse a-synuclein. \u003cstrong\u003eF\u003c/strong\u003e Immunoblot showing various forms of a-synuclein in the TBS, SDS, and urea fraction, using an antibody that detects both human and mouse a-synuclein. The monomeric (lower panel) and higher molecular weight (upper panel) forms are shown separately because the abundant monomeric form was visualized using Pierce ECL Western Blotting Substrate, whereas the less abundant higher molecular weight forms were visualized using the more sensitive SuperSignal West Pico Plus Chemiluminescent Substrate. Note that housekeeping gene loading control is not valid for the blots in \u003cstrong\u003eF\u003c/strong\u003e. \u003cstrong\u003eG\u003c/strong\u003e Relative mRNA expressions of human and mouse a-synuclein, expressed as percentage relative to b-actin mRNA in Control mice. n=4/genotype for all immunoblotting and n=3/genotype for RT-qPCR.Statistical analysis was performed using unpaired t-test (two-tail). ****, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001. ns, nonsignificant.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/54ca93b514ee0acb5ee3377c.jpeg"},{"id":86919374,"identity":"ff714ba9-64e5-416c-8853-0b4fbf03eadb","added_by":"auto","created_at":"2025-07-17 07:22:09","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":517684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of a-synuclein overexpression on M72 and P2 OSNs and their axonal convergence to olfactory bulb glomeruli.\u003c/strong\u003e \u003cstrong\u003eA-D\u003c/strong\u003e Representative (n=16/genotype) whole-mount X-Gal preparations of olfactory epithelium (OE) and olfactory bulb (OB) of Control/M72 (\u003cstrong\u003eA\u003c/strong\u003e) and a-Syn/M72 (\u003cstrong\u003eB\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e) mice. The blue axons in Control/M72 mice (\u003cstrong\u003eA\u003c/strong\u003e) are easily visualized as they exit the epithelium, traverse the cribriform plate, and enter the OB where they converge on two glomeruli in each OB, one on the lateral (red arrow) and one on the medial (red arrowhead) aspect. \u003cstrong\u003eE\u003c/strong\u003e Histogram plots of the number of X-Gal stained OSNs in the entire epithelium of a-Syn/M72 and Control/M72 mice (n=3/genotype). \u003cstrong\u003eF\u003c/strong\u003e Diagrammatic sketch showing the viewing angle of endoturbinates in \u003cstrong\u003eG\u003c/strong\u003e and \u003cstrong\u003eJ\u003c/strong\u003e, and nasal septum in \u003cstrong\u003eH\u003c/strong\u003e and \u003cstrong\u003eK\u003c/strong\u003e. The endoturbinates are marked by Roman numerals, and the ectoturbinates are marked by Arabic numerals. \u003cstrong\u003eG-L\u003c/strong\u003e Representative (n=16/genotype) whole-mount X-Gal preparations of olfactory epithelium and the medial aspect of the OB of Control/P2 (\u003cstrong\u003eG-I\u003c/strong\u003e) and a-Syn/P2 (\u003cstrong\u003eJ-L\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003emice. Axons of blue-stained OSNs in the epithelium Control/P2 mice (\u003cstrong\u003eG\u003c/strong\u003e and \u003cstrong\u003eH\u003c/strong\u003e) converge on a single glomerulus on the medial aspect of the OB. The lateral side of the bulb is not visible in this view. Higher magnification images of the P2 OSNs in Control/P2 and a-Syn/P2 mice are shown in \u003cstrong\u003eI\u003c/strong\u003e and \u003cstrong\u003eL\u003c/strong\u003e, respectively. Axons of P2 OSNs are clearly visible as they emerge from the soma. Due to differences in the distribution patterns of M72 and P2 OSNs in the epithelium, and because M72 and P2 OSNs project to different regions in the OB, whole-mount of M72 mice show dorsal view and those of P2 mice show lateral view. \u003cstrong\u003eM\u003c/strong\u003eHistogram plots of the number of X-Gal stained OSNs in the entire epithelium of a-Syn/P2 and Control/P2 mice (n=3/genotype). Statistical analysis was performed using unpaired t-test (two-tail). ***, \u003cem\u003eP\u003c/em\u003e = 0.0003; **, \u003cem\u003eP\u003c/em\u003e = 0.0095.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/835c2ce813afa5cb01aaff37.jpeg"},{"id":86919386,"identity":"18379277-009e-4f24-aade-54b56ba7f55c","added_by":"auto","created_at":"2025-07-17 07:22:09","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":403636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of a-synuclein overexpression on M72 and P2 glomeruli.\u003c/strong\u003e Higher magnification images of\u003cstrong\u003e \u003c/strong\u003ecoronal section of the olfactory bulbs showing X-Gal stained M72 and P2 glomeruli in Control/M72 (\u003cstrong\u003eA\u003c/strong\u003e), Control/P2 (\u003cstrong\u003eB\u003c/strong\u003e), a-Syn/M72 (\u003cstrong\u003eC\u003c/strong\u003e), and a-Syn/P2 (\u003cstrong\u003eD\u003c/strong\u003e) mice. The sections were counterstained with neutral red to reveal various layers of the bulbs.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/f5d9c1c2225fce99d4384068.jpeg"},{"id":86920816,"identity":"955980b3-edb1-4133-ae3b-018112dd4541","added_by":"auto","created_at":"2025-07-17 07:38:09","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":759318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of a-synuclein overexpression on axonal convergence of M72 and P2 OSNs.\u003c/strong\u003e \u003cstrong\u003eA-D\u003c/strong\u003e Higher magnification images of whole-mount X-Gal preparations of olfactory bulbs of a-Syn/M72 mice, showing supernumerary M72 glomeruli. Individual glomeruli in each bulb are marked with color-coded arrowheads. \u003cstrong\u003eE1-E5\u003c/strong\u003e Representative serial coronal sections of an olfactory bulb from a-Syn/P2 mouse, showing supernumerary M72 glomeruli (color-coded arrowheads). Since P2 glomeruli are located ventrally in the bulb, they are not readily visualized in whole-mount X-Gal preparations.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/01f97658f61df55a2af3d117.jpeg"},{"id":86919375,"identity":"61787a43-4ede-4994-94c4-3a0807d63b28","added_by":"auto","created_at":"2025-07-17 07:22:09","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":360229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOlfactory dysfunction and home cage activity levels in a-Syn mice.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eHistogram showing the retrieval time of a hidden piece of cereal in the buried food pellet retrieval test for general anosmia. \u003cstrong\u003eB\u003c/strong\u003e Time to find an exposed piece of cereal. \u003cstrong\u003eC\u003c/strong\u003e Plot showing sniffing time versus odor trial number for mice habituated to peppermint odor through repeated exposure at 1-minute intervals. On the eighth trial, banana odor (amyl acetate) was introduced to assess discrimination after habituation to the peppermint odor. \u003cstrong\u003eD\u003c/strong\u003eHistogram illustrating the time spent in familiar vs. unfamiliar compartments. \u003cstrong\u003eE\u003c/strong\u003eHistogram illustrating olfactory sensitivity, quantified by sniffing time for mineral oil (diluent) and five different dilutions of amyl acetate. \u003cstrong\u003eF\u003c/strong\u003e Home cage activity data showing animals’ speeds during the dark and light cycles. Average speeds during the dark and light cycles are shown in \u003cstrong\u003eG\u003c/strong\u003e and \u003cstrong\u003eH\u003c/strong\u003e, respectively. \u003cstrong\u003eI\u003c/strong\u003e Home cage activity data showing distance traveled by the mice during the dark and light cycles. Total distances traveled during the dark and light cycles are shown in \u003cstrong\u003eJ\u003c/strong\u003e and \u003cstrong\u003eK\u003c/strong\u003e, respectively. Statistical analysis for \u003cstrong\u003eA\u003c/strong\u003e, \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eJ\u003c/strong\u003e, \u003cstrong\u003eH\u003c/strong\u003e, \u003cstrong\u003eJ\u003c/strong\u003e, and \u003cstrong\u003eK\u003c/strong\u003e was performed using unpaired t-test (two-tail). For \u003cstrong\u003eC\u003c/strong\u003e, statistical analysis was performed separately for trial eight (amyl acetate) using unpaired t-test. For \u003cstrong\u003eD\u003c/strong\u003e, statistical analysis was performed comparing time spent in familiar vs. unfamiliar compartments, separately for a-Syn and Control groups. For all other data (\u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e, and \u003cstrong\u003eI\u003c/strong\u003e), statistical analysis was performed using two-way repeated measures ANOVA with Tukey’s multiple comparison tests. \u003cstrong\u003eE \u003c/strong\u003eRow Factor (Time) F (5, 40) = 43.40, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; Column Factor (Genotype) F (1, 8) = 32.56, \u003cem\u003eP\u003c/em\u003e = 0.0005; Row Factor x Column Factor F (5, 40) = 5.889, \u003cem\u003eP\u003c/em\u003e = 0.0004. \u003cstrong\u003eF\u003c/strong\u003e Row Factor F (4.545, 45.45) = 10.20, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; Column Factor F (1, 10) = 0.6885, \u003cem\u003eP\u003c/em\u003e = 0.4260; Row Factor x Column Factor F (23, 230) = 2.552, \u003cem\u003eP\u003c/em\u003e= 0.0002. \u003cstrong\u003eI\u003c/strong\u003e Row Factor F (4.527, 45.27) = 9.810, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001; Column Factor F (1, 10) = 1.100, \u003cem\u003eP\u003c/em\u003e = 0.3190; Row Factor x Column Factor F (23, 230) = 2.316, \u003cem\u003eP\u003c/em\u003e = 0.0009. *, \u003cem\u003eP\u003c/em\u003e = 0.0361; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0005; ****, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/a448a683afd98846df7f8b05.jpeg"},{"id":102785204,"identity":"b286b518-b0b0-4013-808a-342eb9438e5a","added_by":"auto","created_at":"2026-02-16 16:02:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4889890,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6890617/v1/8a0ac8cd-5079-48e6-9be3-ef83ad6bc176.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eOlfactory sensory map is perturbed in a human wild-type \u003cstrong\u003eα\u003c/strong\u003e-synuclein overexpressing transgenic mouse model of Parkinson’s disease\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSense of smell is often the first casualty of Parkinson\u0026rsquo;s disease (PD) (Fereshtehnejad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and its loss may be both a useful biomarker for disease onset and a clue to pathogenesis. Over 90% of PD patients exhibit olfactory dysfunction, including impairment of odor detection, identification, and/or discrimination (Wu et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This prevalence is even higher than that of the cardinal sign of resting tremor (~\u0026thinsp;75%). Furthermore, the association reported between impaired olfaction and subsequent development of PD (G. W. Ross et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), suggests that it is a very early sign of idiopathic PD (Haehner et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In many patients, olfactory loss predates the development of clinical PD by more than 20 years (Fereshtehnejad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). There is strong evidence that α-synucleinopathy, one of the hallmarks of PD, first appears in the olfactory system, followed by divergent spread of the pathology, either up to the limbic cortex or down to the lower brainstem (Adler \u0026amp; Beach, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Beach et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Olfactory bulb α-synucleinopathy has a high sensitivity (95%) and specificity (91%) for PD, accurately predicting the presence of α-synuclein deposits in other brain regions (Beach et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Indeed, among PD\u0026rsquo;s prodromal non-motor manifestations, olfactory loss has the highest accuracy for diagnostic performance and is one of the three prodromal markers (the other two being REM sleep behavior disorder and constipation) for which the ability to predict conversion to PD is supported by the strongest evidence (Postuma \u0026amp; Berg, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, recent data from the Baltimore Longitudinal Study of Aging provide new insights into a relationship between olfaction and motor function in older adults (Tian et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This study revealed an association between higher olfactory function and slower decline in manual dexterity and psychomotor speed in participants aged 50 years or older (Tian et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The severity of olfactory dysfunction in PD increases with disease progression (Berendse et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Roos et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), and severe hyposmia is one of the early predictors of PD mortality (Backstrom et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Yet the mechanism of this olfactory loss and the cause-and-effect relationship between olfactory dysfunction and α-synucleinopathy, as well as their linkage to dopaminergic neurodegeneration and motor symptoms in PD, are unknown. This major gap in our understanding of pathogenesis and disease progression represents a barrier to the development of strategies for more efficacious treatments.\u003c/p\u003e\u003cp\u003eIn the olfactory system, detection and discrimination of myriad odorants are achieved by approximately 1,000 discrete odor receptors, each expressed by different subsets of olfactory sensory neuron (OSNs) embedded within the olfactory neuroepithelium that lines the nose (Buck \u0026amp; Axel, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Chess et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Ngai et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Ressler et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). A given OSN sends 10 to 30 receptor-bearing cilia from its dendrite into the olfactory mucus and extends a long non-myelinated axon to the ipsilateral olfactory bulb. OSNs expressing the same odor receptor project to a common glomerulus in a spatially conserved region of the bulb, synapsing with mitral and tufted cell dendrites and thereby forming a topographic olfactory sensory map that supports the detection and discrimination of particular odors (Mombaerts et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Ressler et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Vassar et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). It is not yet known whether the olfactory sensory map undergoes degeneration in PD, thereby contributing to olfactory dysfunction. Recent studies (Kim et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Luk et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Rey et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Uemura et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) have clearly shown that that inoculation of α-synuclein preformed fibrils leads to cell-to-cell prion-like transmission of pathologic α-synuclein in anatomically interconnected regions, suggesting that connectivity is a determinant of susceptibility. Since axons of OSNs, the peripheral components of the stereotypic olfactory sensory map, synapse mainly with mitral cells, which harbor α-synuclein deposits in PD (Del et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), OSNs are highly vulnerable, making them a potential source of prodromal biomarkers. OSNs residing in the peripheral nasal epithelium may especially warrant detailed analysis, since they are developmentally related to the central nervous system, yet are accessible for diagnosis by relatively non-invasive biopsy or nasal brushing. Thus, to understand the affect of olfactory bulb α-synuclein pathology on the olfactory sensory map, we generated two double-transgenic mice that express either tagged-M72 or tagged-P2 odor receptor in a human α-synuclein over-expressing background, enabling the first demonstration of degenerative changes in the olfactory sensory map. We discuss the implications of these findings for human PD.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003eThe olfactory sensory map\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOlfactory processing begins when odorant molecules bind to odorant receptors on the cilia of OSNs within the olfactory neuroepithelium lining the nasal cavity. The transduced odor signals are then relayed to the olfactory bulb. From there, the encoded information is transmitted to the olfactory cortices and subsequently to various other brain regions (Buck, 1996; Shepherd, 1994). In mice, a mature OSN typically expresses one allele (Chess et al., 1994) of one (Malnic et al., 1999) of the 1,141 discrete odorant receptor genes (Barnes et al., 2020). A given OSN sends 10 to 30 receptor-bearing cilia from its dendrite into the olfactory mucus and extends a long non-myelinated axon through the epithelium and cribriform plate to the ipsilateral olfactory bulb. OSNs expressing the same odorant receptor protein (represented by individual colors in Fig. 1) are scattered in highly overlapping and strikingly complex zones in the olfactory neuroepithelium (Zapiec \u0026amp; Mombaerts, 2020). However, they converge onto one or a few glomeruli in a spatially conserved region of the ipsilateral olfactory bulb (Bozza et al., 2002; Mombaerts et al., 1996; Ressler et al., 1994; Vassar et al., 1994), where they synapse with the apical dendrites of mitral cells and tufted cells, forming an olfactory sensory map that enables the detection and discrimination of specific odors. The axons of mitral cells project to a variety of more central and higher-order brain regions that mediate learned and innate odor-driven behaviors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression of human\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e-synuclein in the olfactory system of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e-Syn transgenic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigures 2-4 combine data from a-Syn transgenic, a-Syn/M72 and a-Syn/P2 double-transgenic, and their littermate control mice. Since there is no difference either in the pattern or levels of a-synuclein among these mice, for simplicity, the mice are represented as a-Syn and Control. The pattern of expression of the transgene, human a-synuclein, in the olfactory system was assessed by immunofluorescence using antibodies specific to human a-synuclein protein. No human a-synuclein immunofluorescence was detected in the olfactory epithelium that lines any of the endoturbinates or ectoturbinates in the nasal cavity of either a-Syn or Control mice. Representative images of endoturbinate I are shown in Fig. 2. \u0026nbsp;In the olfactory bulb of a-Syn mice, intense human a-synuclein immunofluorescence was detected in all layers except the superficial olfactory nerve layer that is comprised primarily of axons of the olfactory sensory neurons (Fig. 3). The immunofluorescence was localized primarily to the neuronal processes. However, the soma of some mitral cells showed intense human a-synuclein immunofluorescence. As expected, no immunofluorescence to human a-synuclein was observed in age-matched Controls (Fig. 3H). We also confirmed the presence of human a-synuclein in the olfactory bulbs of a-Syn mice using immunoblotting. A single band at ~15 kDa, corresponding to monomeric human a-synuclein, was detected in a-Syn mice, whereas no human a-synuclein was detected in Control mice (Fig. 4A). \u0026nbsp;Since the majority of pathological a-synuclein is phosphorylated at serine 129 (pSer129), we also performed immunoblotting using antibodies to pSer129-a-synuclein. As was the case for monomeric human a-synuclein, pSer129-a-synuclein was detected only in a-Syn mice (Fig. 4A). Since the antibody specific to human a-synuclein detects only monomeric a-synuclein, we also used a second antibody that binds to both human and mouse a-synuclein and detects all forms of a-synuclein. Use of this antibody revealed, in addition to monomeric a-synuclein at ~15 kDa, a variety of forms of a-synuclein \u0026mdash; ranging from 30kDa to over 250 kDa \u0026mdash; in a-Syn mice. \u0026nbsp;However, in the Control mice only monomeric a-synuclein was detected, albeit unsurprisingly to a lesser level (Fig. 4E). In PD, a-synuclein undergoes conformational changes to form toxic insoluble aggregates, a neuropathological feature that is associated with disease progression. To assess the accumulation of abnormal a-synuclein aggregates in the olfactory bulb, TBS-soluble and TBS-insoluble (soluble in SDS and urea) fractions of a-synuclein were collected through sequential extraction with high-speed ultracentrifugation (Bandopadhyay, 2016) followed by immunoblotting using the antibody that binds to both human and mouse a-synuclein. As expected, monomeric a-synuclein was present in the TBS-soluble fraction from both a-Syn and in a smaller amount from Control mice (Fig. 4F). However, the other higher molecular weight forms \u0026mdash; ranging from 30kDa to 250 kDa \u0026mdash; were present only in the TBS-soluble fraction from a-Syn mice. Similarly, various forms of a-synuclein in the SDS- and urea-soluble fractions were detected only in a-Syn mice (Fig. 4F). It has been suggested that the SDS solubilizes a-synuclein oligomers that can include membrane bound forms, whereas urea denatures the insoluble aggregated and fibrillar or amyloidogenic forms of a-synuclein (Anderson et al., 2006; Bandopadhyay, 2016). Lastly, we assessed expression levels of both human and mouse a-synuclein mRNA in the olfactory bulb using RT-qPCR. Whereas human a-synuclein mRNA was detected only in a-Syn mice, mouse a-synuclein mRNA was detected, in similar levels, in both a-Syn and Control mice (Fig. 4G), indicating that the overexpression of human a-synuclein mRNA had no impact on the levels of endogenous mouse a-synuclein mRNA in a-Syn mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlteration of olfactory sensory map in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e-Syn/M72 and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e-Syn/P2 double transgenic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the effect of a-synuclein overexpression on the olfactory sensory map, 12-month-old a-Syn/M72 and P2 and littermate Control/M72 and P2 mice were processed for whole-mount X-Gal staining (n=16/genotype). In the Control/M72 mice, the M72 olfactory sensory neurons (OSNs) were readily visualized by blue staining, and their axons converge on two glomeruli, one on the lateral and one on the medial aspect of each olfactory bulb (Fig. 5A). a-Syn/M72 mice exhibited a drastic reduction in the number of M72 OSNs. Consequently, OSN axonal projections to the bulb were dramatically reduced (Fig. 5B-E), leading to formation of small glomeruli (Fig. 6C). While the OSN axonal projection patterns of all a-Syn/M72 showed changes in both olfactory bulbs (Fig. 5B, C, D), many mice showed disproportionate unilateral changes (Fig. 5C), which is highly intriguing since cardinal motor symptoms of PD are usually unilateral at the earliest stage. As seen for the a-Syn/M72 mice, P2 OSNs and their axons were dramatically reduced in the a-Syn/P2 mice (Fig. 5J-M). Consequently, the P2 glomeruli in a-Syn/P2 mice were smaller and often their staining intensity was lighter, compared to Control/P2 mice (Fig. 6B, D). Although the M72 and P2 OSNs in a-Syn/M72 and P2 mice project broadly to similar locations in the bulb as their counterparts in Control mice, unlike the tight glomerular organization in Controls (two glomeruli per bulb \u0026mdash; one lateral and one medial), supernumerary glomeruli of varying size were common in the a-Syn/M72 and a-Syn/P2 mice (Fig. 7 and Table 1). The positions of individual glomeruli (e.g. M72 or P2 glomeruli) have been shown to be topographically defined. However, there was no consistency between individual mice in the location of these supernumerary glomeruli (Fig. 7), indicating that a-synuclein overexpression affects the mechanism guiding the convergence of axons of OSNs expressing a particular odorant receptor.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenotype\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(n=10/genotype)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGlomeruli/bulb\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e(Min\u0026mdash;Max)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003eM72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e2.1 (2\u0026mdash;3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003ea-Syn/M72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e4.3 (3\u0026mdash;6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003eP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e2 (2\u0026mdash;2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 122px;\"\u003e\n \u003cp\u003ea-Syn/M72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e3.4 (3\u0026mdash;4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable. 1:\u0026nbsp;\u003c/strong\u003eNumber of odorant receptor-specific glomeruli per olfactory bulb. n=20 bulbs from 10 mice per genotype.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOlfactory dysfunction in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e-Syn/M72 and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ea\u003c/strong\u003e\u003cstrong\u003e-Syn/P2 double transgenic mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human wild-type\u0026nbsp;a-synuclein overexpressing transgenic mice have previously been shown to exhibit olfactory dysfunction (Biju et al., 2025; Fleming et al., 2008). To confirm olfactory dysfunction in the double transgenic mice,\u0026nbsp;a-Syn/M72 and\u0026nbsp;a-Syn/P2 (together represented as\u0026nbsp;a-Syn mice in Fig. 8), and their respective littermate controls (n=5/genotype) were subjected to a battery of olfactory behavioral tests. The\u0026nbsp;a-Syn mice took significantly more time than Control mice to retrieve hidden cereal (Fig. 8A), a measure of hyposmia. The time to find an exposed piece of cereal was not significantly different between\u0026nbsp;a-Syn and Control mice (Fig. 8B). In test for the ability to discriminate between two odors using a habituation/dishabituation paradigm, the\u0026nbsp;a-Syn mice exhibited deficits in odor discrimination (Fig. 8C), seen as decreased exploratory time, compared with Control mice, upon presentation of a second odor following habituation to a first odor. We also used a paradigm based on the time spent in familiar versus unfamiliar compartments to assess olfactory dysfunction. This task relies on rodents typically preferring locations marked with their own scent (familiar compartment). Consequently, mice with normal olfaction will spend more time in a familiar compartment when choosing between familiar and unfamiliar compartments. The test further confirmed olfactory dysfunction in\u0026nbsp;a-Syn mice, as indicated by the equal amounts of time spent in familiar and non-familiar compartments (lack of preference for familiar compartment; Fig. 8D). Since the number of M72 and P2 OSNs was dramatically lower in the odorant receptor tagged double transgenic mice, we also assessed olfactory sensitivity. While the lowest dilution of the odorant at which the exploratory time of Control mice was significantly different compared with mineral oil (diluent) was 10\u003csup\u003e-4\u003c/sup\u003e, the lowest dilution for\u0026nbsp;a-Syn mice was 10\u003csup\u003e-2\u003c/sup\u003e, indicating a drastically reduced olfactory sensitivity in a-Syn mice (Fig. 8E). Since olfactory behavioral tests are influenced by the overall activity levels of the animals, and since a-Syn mice exhibit hyperactivity at younger ages, we assessed the general activity levels (speed and distance travelled) of the mice in their home cages. Although there was a temporal difference in activity levels between a-Syn and Control mice during the dark cycle (Fig 8F, I), total activity levels were similar during both the dark (Fig 8G, J) and light (Fig. 8H, K) cycles (when the olfactory tests were performed).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWe generated two separate odorant receptor-tagged mice in a human α-synuclein over-expressing background (Line 61) and showed that there was a dramatic decrease in the number of OSNs and perturbations in the stereotypic convergence of their axons in the olfactory bulbs, suggesting that neurodegenerative changes in the olfactory sensory map contribute to olfactory dysfunction in PD. Except for a recent study that computed the total number of OSNs in an aged human specimen (n of 1) to be ~\u0026thinsp;2.7\u0026nbsp;million (Low et al., 2024), to our knowledge, no data exist to assess whether there is a reduction in the number of OSNs in PD patients. This lack of data is unsurprising given the technical challenges associated with assessing the total number of OSNs in humans, especially given that the olfactory epithelium in humans is patchy and interspersed with respiratory epithelium (Holbrook et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, our extrapolation, based on the number of OSN in mouse olfactory epithelium and on glomerular data in human PD patients, predicts that the number of OSNs in PD patients is likely to be dramatically lower than that of healthy subjects. For example, analysis of human olfactory bulbs revealed that global glomerular voxel volume of olfactory bulbs in PD patients is half that of control patients (Zapiec et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This study further showed that the higher the α-synuclein load in the olfactory bulb, the lower the global glomerular voxel volume, suggesting a causal relationship between α-synuclein pathology and glomerular voxel volume. In studies of odorant receptor-tagged mice, researchers have demonstrated a strong linear correlation between OSN number and glomerular voxel volume (Bressel et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Based on this, it has been suggested that glomerular voxel volume can be used as a surrogate marker for the number of OSNs (Bressel et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Together, these reports suggest that the significant reduction in global glomerular voxel volume in the olfactory bulb of PD patients corresponds to a significant reduction in the number of OSNs in the nasal epithelium, as seen in α-Syn/M72 and α-Syn/P2 mice in the current study. Thus, OSNs from PD patients warrant in-depth analysis for transcriptomic and metabolomic signatures, as it could provide crucial insight into the pathogenesis of PD.\u003c/p\u003e\u003cp\u003eAn important question is to what extent are our data on M72 and P2 OSNs relevant to OSNs expressing other odorant receptors in the nasal epithelium of PD patients. There are between 4 to 8\u0026nbsp;million total OSNs in the olfactory epithelium of an adult mouse, and each of their olfactory bulbs contains about 3,600 to 3900 glomeruli (Richard et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), making it technically challenging to assess subtle neuroanatomical changes. However, distributed in two non-overlapping zones (Zolfr160 and Zolfr17) in the olfactory epithelium (Zapiec \u0026amp; Mombaerts, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), the absolute number of M72- and P2-expressing OSNs in a young adult mouse is only about 6,000 and 12,000, respectively, and typically, they each project to only two glomeruli in each bulb (Biju et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Bressel et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Because only a small proportion of the total OSNs express either M72 or P2 odorant receptors, and they project to only two glomeruli each, it may even be possible to reveal subtle neuroanatomical olfactory system changes, thereby facilitating precise definition of the structural changes in the olfactory epithelium and olfactory bulb that lead to impaired sense of smell in Parkinson\u0026rsquo;s disease. This theory is based on the hypothesis that olfactory dysfunction in PD affects all odors, and thus a global change in OSN number and projection pattern will be faithfully reflected by the M72 and P2 OSNs. Indeed, Doty et al. first observed that PD features a general olfactory deficit, rather than a deficit to certain specific odors (Doty et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Subsequently, there were reports showing the opposite \u0026mdash; that PD patients experience reduced sensitivity to certain specific odors such as banana, pineapple, pizza, licorice, dill pickle, cinnamon, wintergreen, smoke, and gasoline (Bohnen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Double et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hawkes \u0026amp; Shephard, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). However, more recent studies (Chou \u0026amp; Bohnen, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Doty, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hahner et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Morley et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Vaswani et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) have confirmed the original findings of Doty et al. (Doty et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) that olfactory dysfunction in PD is not restricted to specific odors. In fact, a recent study that used eight machine learning models to distinguish PD hyposmia from non-PD hyposmia showed that PD-related hyposmia does not exhibit a unique pattern of odor selectivity distinct from general hyposmia (Mitchell et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Together, these studies clearly establish that olfactory dysfunction is unrelated to specific odorants; however, it is possible that the extent of damage may vary among OSNs expressing a particular odorant receptor. For example, we found that 76% M72 OSNs were lost in the α-Syn/M72, whereas only 45% of P2 OSNs were lost in the α-Syn/P2. The exact reason for this differential loss is unclear. One possibility is that axons of M72 OSNs need to travel longer distances to reach their glomeruli in the bulb, making these unmyelinated axons more vulnerable to α-synuclein induced toxicity.\u003c/p\u003e\u003cp\u003eSense of smell is overwhelmingly the first casualty of PD, often predating the development of clinical PD by over 20 years (Fereshtehnejad et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); and since olfactory dysfunction is likely an early victim of the same pathological mechanism that eventually leads to PD\u0026rsquo;s motor symptoms, it may be a window into pathogenesis. However, a major roadblock to understanding olfactory dysfunction in human prodromal PD patients is the very fact that there is currently no way to identify prodromal patients for such studies. Moreover, the molecular pathology in post-mortem human brain samples may no longer reflect what occurred in the prodromal stage of PD. However, the mechanism of PD-related olfactory deficits can be effectively explored through the use of mouse models, since the neural mechanisms of odor processing in rodents are well understood and share important characteristics with humans. To best represent prodromal PD, we used 12-month-old mice for all studies, for these reasons: (1) Age is the greatest risk factor for PD, and it is conceivable that young mice are capable of mounting a sufficient defense against toxic insults (Borghammer \u0026amp; Van Den Berge, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). (2) According to The Jackson Laboratory, 12 months in mice is the equivalent of 45 years in humans; since the average age of clinical diagnosis of PD is 65, and olfactory loss can occur over 20 years prior to clinical diagnosis, 45 can be considered reasonably within the prodromal period. (3) Twelve months of age in α-Syn mice is considered a prodromal stage (Richter et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). While there are reports of α-Syn mice exhibiting \u0026ldquo;motor deficits\u0026rdquo; at earlier ages (Rabl et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), these \u0026ldquo;motor deficits\u0026rdquo; are actually deficits in motor learning, motor coordination or both and should not be confused with the typical motor symptoms of clinical PD. The term \u0026ldquo;motor dysfunction\u0026rdquo; includes: (a) deficits in motor learning, (b) deficits in fine motor skills/motor coordination, and (c) gross deficits in ambulation/gait. Depletion of striatal dopamine alone is sufficient to induce deficits in motor learning and fine motor skills/motor coordination; consequently, these deficits (e.g., in handwriting, dexterity, etc.) are evident in the prodromal stage. However, the gross motor deficit in ambulation/gait that patients exhibit at the time of clinical diagnosis require depletion of both striatal and nigral dopamine (Gonzalez-Rodriguez et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, α-Syn mice exhibit striatal dopamine loss and deficit in motor learning/fine motor tasks during motor coordination tests; however, nigral dopaminergic neuronal loss and gait dysfunctions are not observed in α-Syn mice at 12 months of age, which is why they are considered a prodromal model of PD.\u003c/p\u003e\u003cp\u003eThe exact mechanism for the observed changes in the olfactory sensory map in our double-transgenic mice is unclear. OSNs continuously regenerate, and the newly formed neurons project their axons to the olfactory bulb throughout the lifespan of the animal (Brann \u0026amp; Firestein, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, connections to the olfactory bulb must be reestablished continuously to maintain a precise olfactory sensory map. Our data show a dramatic reduction in the number of OSN and supernumerary glomeruli, indicating that both regeneration of OSNs and targeting of their axons are affected. It has been previously shown that both the peripheral development and target destination of OSNs are influenced by the activity of mitral cells in the olfactory bulb (Biju et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In mice having a gene-targeted deletion of the Shaker potassium ion channel (Kv1.3), mitral cells have altered action potential and increased firing frequency (Fadool et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). M72 and P2 mice in Kv1.3-null background also exhibit a drastic reduction in OSN numbers, as well as supernumerary glomeruli (Biju et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Indeed, mitral cells overexpress human α-synuclein in our α-Syn/M72 or P2 double transgenic mice. α-Synuclein is found in axon terminals and plays an active role in the downregulation of neurotransmitter release (Nemani et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thus, overexpression of human α-synuclein could disrupt mitral cell activity, causing perturbations in the olfactory sensory map as seen in the Kv1.3-null. Of note, over-expression of normal α-synuclein (vs. mutant form) is sufficient to cause PD, and a gene-dosage effect exists in that triplication results in higher α-synuclein levels, more severe pathology, and earlier onset than duplication (Fuchs et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; O. A. Ross et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Singleton et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Although we focused on the olfactory system, α-Syn mice harbor α-synuclein pathology in many other brain areas (Rockenstein et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) as seen in PD. Therefore, we cannot completely exclude potential contributions of extra-olfactory bulb α-synuclein pathology on the observed effect in the olfactory sensory map.\u003c/p\u003e\u003cp\u003eIn summary, we provide compelling evidence for perturbations in the olfactory sensory map of a human wild-type a-synuclein overexpressing transgenic mouse model of PD, and for their relevance to understanding olfactory dysfunction in PD. If olfactory dysfunction in PD is accompanied by (whether or not due to) degenerative changes in the topographic olfactory sensory map, these early structural/cellular changes should be accompanied by the appearance of disease-specific changes in the molecular atlas of the OSNs in the nasal epithelium. Mapping of this molecular atlas would not only yield minimally invasive biomarkers for prodromal prognostication of the disease, but may also lead to insights into both disease pathogenesis and disease progression, which in turn could help development of new neuroprotective strategies.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eMice\u003c/h2\u003e\u003cp\u003eThree different kinds of transgenic mice in C57BL/6J background were used: (1) mThy1-hSNCA (Line 61), (2) M72-IRES-tau-LacZ, and (3) P2-IRES-tau-LacZ. The mThy1-hSNCA mice (henceforth, α-Syn mice) express the human wild-type α-synuclein gene under the direction of the mouse thymus cell antigen 1 (thy1) promoter (Rockenstein et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These mice are useful for studying PD pathogenesis and neurodegeneration elicited by the toxic effects of aggregation and somatic accumulation of human α-synuclein. The mThy1-hSNCA mice were a generous gift from Dr. Randy Strong (University of Texas Health Science Center at San Antonio, TX). M72-IRES-tau-LacZ and P2-IRES-tau-LacZ mice (henceforth, M72 and P2 mice) were generated previously (Mombaerts et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Zheng et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) via placement of an internal ribosome entry site (IRES) directing the translation of tau-lacZ fusion protein immediately downstream of the M72 (Olfr160) or P2 (Olfr17) odorant receptor stop codon. In these genetically altered strains of mice, olfactory sensory neurons that transcribe the modified M72 or P2 receptor allele also express tau-lacZ in their cell bodies and axons, enabling direct visualization of individual olfactory sensory neurons in the epithelium, as well as the pattern of projections of these neurons in the olfactory bulb, by histological staining for β-galactosidase (X-Gal) activity. The M72 and P2 mice were a generous gift from Dr. Debra Ann Fadool (Florida State University, FL). We generated double-transgenic M72 and P2 odorant receptor-tagged mice that over-express human wild-type α-synuclein (α-Syn/M72 or α-Syn/P2 mice) by breeding homozygous M72 and P2 mice with hemizygous α-Syn mice. All the double-transgenic mice used in this study were hemizygous for human wild-type α-synuclein and homozygous for the modified M72 or P2 receptor allele. Since the transgene (human wild-type α-synuclein) in α-Syn mice is inserted into the X chromosome, female mice exhibit diminished and variable PD deficits (Biju et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Chesselet et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Grant et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), likely due to random inactivation of the transgene-bearing chromosome in hemizygous females during early embryogenesis. Therefore, we used male α-Syn/M72 and α-Syn/P2 mice, at 12 months of age, for all our studies. The mice were housed in standard plexiglass cages with 7099 TEK-Fresh animal bedding (Envigo, Indianapolis, IN) and were fed a standard Teklad irradiated LM-485 mouse diet (Cat # 7912; Envigo, Indianapolis, IN) \u003cem\u003ead libitum\u003c/em\u003e and with full-time access to acidified drinking water. The mice were maintained in a 12/12 hour light/dark cycle at 24\u003csup\u003eO\u003c/sup\u003eC room temperature and 50\u0026ndash;55% humidity. Animal husbandry was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Society for Neuroscience Policies on the Use of Animals and Humans in Research. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the South Texas Veterans Health Care System, Audie L. Murphy Division, and the University of Texas Health Science Center, San Antonio. The smallest possible number of mice was used (based on power calculations) and all efforts were made to minimize suffering. All experiments were randomized and performed blind-coded whenever feasible.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eOlfactory behavioral tests\u003c/h3\u003e\n\u003cp\u003eThe olfactory behavioral tests were performed in a dedicated rodent behavior-testing room (sound attenuated and with HEPA-filtered air) during the light cycle with 30\u0026ndash;40 lux light intensity and 24\u003csup\u003e0\u003c/sup\u003eC room temperature maintained throughout the tests. General anosmia was assessed as described before (Biju et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Biju et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) by recording latency to retrieval of a cereal hidden under the bedding. Olfactory discrimination was assessed by either recording of exploratory time upon presentation of a second odor (amyl acetate \u0026mdash; banana flavor) following habituation to a first odor (peppermint) or by recording the time spent in familiar vs. non-familiar compartments using our published protocol (Biju et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Olfactory sensitivity was assessed by recording exploratory time to one of five dilutions of odorant (10\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e, 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e, and 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e dilutions of amyl acetate in mineral oil) for one 3-minute session using a published method (Witt et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Briefly, the mice were acclimated individually to a clean standard mouse cage (without bedding) with a piece of filter paper enclosed in a tissue cassette for four sessions of 15 minutes each. Immediately after the acclimation the mice were introduced to the test cage with a tissue cassette containing a filter paper dampened with equal volumes of either mineral oil or one of the five dilutions of amyl acetate, and the sniffing time was recorded in a three-minute session. After completing the initial three-minute session, for all mice were immediately started on the second three-minute session with the next dilution of odorant, chosen randomly, and so on until all the five dilutions of amyl acetate and mineral oil were completed.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eHome cage activity monitoring\u003c/h2\u003e\u003cp\u003eGeneral activity in the home cage was recorded using the ActiMot2 infrared light beam activity monitoring system (25 beams each on X and Z directions and 16 beams on Y direction; TSE Systems, Inc., Chesterfield, MO) using our published protocol (Biju et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The home activity of the mice was recorded at a sampling rate of 100 Hz with a recording interval of ten minutes for 24 hours continuously. Speed and total distance travelled during the light and dark phases were analyzed and compared among the groups.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\u003cp\u003eThe mice were anesthetized with an overdose of ketamine HCl/xylazine HCl solution and perfused transcardially with 10\u0026ndash;20 ml ice-cold phosphate-buffered saline (PBS, pH 7.4) followed by an equal volume of ice-cold 4% paraformaldehyde in PBS. Brains were removed and post-fixed overnight in the same fixative at 4\u003csup\u003e0\u003c/sup\u003eC. Parts of the heads containing nasal epithelium were first post-fixed overnight in the same fixative and then decalcified in 0.3 M EDTA for 48 hours at 4\u003csup\u003e0\u003c/sup\u003eC. The tissues were cryoprotected in sequential solutions of sucrose (10% for 2 h, 20% for 2 h, and 30% for 24\u0026ndash;48 h), and then embedded in Tissue-Tek OCT compound. Cryosections of olfactory epithelia and bulbs were prepared at 15 \u0026micro;m thickness in the coronal plane using a Leica CM 1950 cryostat. Immunofluorescence was performed as described (Biju et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Briefly, the sections were incubated with an antibody (1:2,000 dilution) specific for human a-synuclein (cat # ab138501; abcam, Waltham, MA) overnight at 4\u003csup\u003e0\u003c/sup\u003eC. The secondary antibodies were conjugated with Alexa Fluor\u0026reg; 488 (1:200; Molecular Probes). Sections were coverslipped with VECTASHIELD Vibrance\u0026reg; Antifade Mounting Medium with DAPI (cat # H-1800; Vector Laboratories, Inc., Newark, CA). Fluorescent images were analyzed with a Keyence BZ-X800 microscope. Stringent control procedures were utilized to ensure specificity of immunofluorescence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eX-Gal staining\u003c/h2\u003e\u003cp\u003eThe mice were anesthetized and then perfused transcardially as described above for immunofluorescence. The heads were removed, deskinned and post-fixed overnight in the fixative at 4\u003csup\u003e0\u003c/sup\u003eC. The reagents and protocol for X-Gal staining have been described (Biju et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Mombaerts et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Briefly, the heads were decalcified in 0.3 M EDTA for 3 days at 4\u003csup\u003e0\u003c/sup\u003eC and then the nasal epithelia and olfactory bulbs were exposed by removing the skull from the dorsal side. The heads were washed with Buffer A for 5 minutes and then again for 25 minutes. The heads were then incubated twice with Buffer B for 5 minutes each, followed by an 8-hour incubation in Buffer C. Following a 10-minute fixation in 4% paraformaldehyde and then a wash in PBS, whole mount images were captured using a stereomicroscope. Thereafter, parts of the heads containing nasal epithelium and olfactory bulbs were cryoprotected in 30% sucrose, frozen in OCT and then cryosectioned at 30 \u0026micro;m thickness. The cryosections were counterstained with neutral red and used for manual counting of M72 and P2 olfactory sensory neurons (Biju et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eImmunoblotting\u003c/h2\u003e\u003cp\u003eImmunoblot analysis of a-synuclein in the olfactory bulb was performed using our published protocol (Biju et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Briefly, following euthanasia by CO\u003csub\u003e2\u003c/sub\u003e inhalation, the mice were decapitated. The olfactory bulbs were quickly dissected out, snap frozen in liquid nitrogen, and then processed for protein extraction. Protein concentrations were determined through Bradford assay. Samples (20 mg) were loaded on Invitrogen Novex 12% Tris-Glycine Plus Midi Gels (cat # WXP01226BOX; ThermoFisher Scientific, Waltham, MA), electrophoresed for 50 minutes at 150 volts, and then transferred onto nitrocellulose membranes. The membranes were stained with Ponceau S to confirm equal loading. The membranes were blocked with 5% nonfat dried milk in 1X TBST buffer and then incubated overnight with one of the following antibodies, prepared in 5% nonfat dried milk, at 4\u003csup\u003e0\u003c/sup\u003eC: (1) human a-synuclein (cat # ab138501; abcam, Waltham, MA), (2) human a-synuclein phosphorylated at serine 129 (cat # ab168381; abcam), (3) human and mouse a-synuclein (cat # ab212184; abcam), (4) GAPDH (cat # G9545; Sigma-Aldrich, Inc. St. Louis, MO), or (5) b-actin (JLA20; cat # AB_528068; DSHB, University of Iowa). The bands were visualized using Pierce ECL Western Blotting Substrate (Cat # 32209; ThermoFisher Scientific). Optical density measurements of the bands were performed using NIH ImageJ software. Analysis of levels of soluble and insoluble a-synuclein in the olfactory bulb was performed by sequential extraction of proteins in Tris-buffered saline (TBS), sodium dodecyl sulphate (SDS) buffer, and urea buffer using a published method (Bandopadhyay, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Subsequent immunoblot analysis of the TBS, SDS, and urea fractions of the proteins was performed as described above; however, the protein bands were visualized using SuperSignal West Pico Plus Chemiluminescent Substrate (cat # 34580; ThermoFisher Scientific).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eReverse transcription-quantitative real-time PCR (RT-qPCR) analysis\u003c/h2\u003e\u003cp\u003eRNA from snap-frozen olfactory bulbs was isolated using TRIzol/chloroform extraction and RNeasy Mini Kit (cat# 74106; QIAGEN Science, Germantown, MD) (Biju et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). RNA was converted into cDNA using the SuperScript VILO cDNA Synthesis Kit (cat# 11754250; Invitrogen/ThermoFisher Scientific) and qPCR was performed using pre-designed TaqMan probes (ThermoFisher Scientific) for human a-synuclein (Hs00240906_m1), mouse a-synuclein (Mm01188700_m1) and b-actin (Mm02619580_g1) on QuantStudio 3 (ThermoFisher Scientific). Transcriptional levels were determined using ∆∆Ct method, and mRNA percentage relative to b-actin in Control mice was calculated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eStatistical analyses of the data were performed in R version 3+ (Vienna, Austria) or GraphPad Prism 10 (GraphPad Software, Inc.). As specified in the legend for each figure, either independent unpaired t-tests or two-way repeated measures ANOVA were used. All F values and degrees of freedom used for ANOVA are also specified in the legends of respective figures. All sets of continuous data were tested for normality using the Shapiro-Wilk test. Results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and differences in mean were considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eDATA AVAILABILITY\u003c/h2\u003e\u003cp\u003eAll raw data and any additional information on methodology or data analyses used in the current study will be available upon request to the corresponding authors.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e\u003cp\u003eAll authors declare that they have no financial or non-financial competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eRAC and KCB conceived and designed the study. KCB, ETH, ACF-O, and AMS performed the behavioral tests, X-Gal staining, histology, quantification of OSNs, and RT-qPCR. SKH, AMS, and ETH analyzed the immunoblot and behavioral data. ETH, KCB, and SKH designed and performed the statistical analysis. All authors were involved in overall data analysis. KCB and RAC wrote the manuscript. All authors read, commented, and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eThis work was supported by a Merit Review Grant from the Veterans Health Administration (1 I01 BX 003157) and a grant from the William and Ella Owens Medical Research Foundation, both awarded to RAC, as well as pilot and career development grants from the Perry \u0026amp; Ruby Stevens Parkinson\u0026rsquo;s Disease Center of Excellence, awarded to KCB. We thank Dr. Debra Ann Fadool at Florida State University and Dr. Randy Strong at UTHSCSA for providing the original breeding pairs of M72/P2 odorant receptor tagged and a-synuclein transgenic mice, respectively.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdler, C. H., \u0026amp; Beach, T. G. (2016). Neuropathological basis of nonmotor manifestations of Parkinson's disease. \u003cem\u003eMov Disord\u003c/em\u003e, \u003cem\u003e31\u003c/em\u003e(8), 1114\u0026ndash;1119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mds.26605\u003c/span\u003e\u003cspan address=\"10.1002/mds.26605\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAnderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J., Kling, K., Lee, M., Diep, L., Keim, P. 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Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit. \u003cem\u003eNeuron\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(1), 81\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/pubmed/10798394\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/pubmed/10798394\" targettype=\"URL\" 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":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-6890617/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6890617/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOlfactory dysfunction, often the earliest symptom of Parkinson\u0026rsquo;s disease (PD), can precede clinical diagnosis by over 20 years, yet its mechanism and link to a-synuclein pathology remain unclear. To understand the impact of α-synuclein pathology on the topographic olfactory sensory map that supports the detection and discrimination of particular odors, we created two double transgenic mouse models (a-Syn/M72 and a-Syn/P2) expressing tagged-M72 or tagged-P2 odor receptors in a human wild-type α-synuclein over-expressing background. We demonstrated that the sensory map is disrupted in these mice. Histological analysis showed a significant reduction in M72 and P2 olfactory sensory neurons (OSNs), with altered glomerular topographies as axons converged into supernumerary glomeruli of varying size and location. These findings suggest that a-synuclein overexpression impairs the mechanism guiding the convergence of OSN axons and thus formation of a precise olfactory sensory map. As OSNs in the nasal epithelium are accessible via non-invasive biopsy, they are a potential source of prodromal PD biomarkers.\u003c/p\u003e","manuscriptTitle":"Olfactory sensory map is perturbed in a human wild-type α-synuclein overexpressing transgenic mouse model of Parkinson’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 07:22:04","doi":"10.21203/rs.3.rs-6890617/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-13T12:38:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"270278841763421254794656050658748942149","date":"2025-08-12T13:50:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"176226037589286654898599421668514285362","date":"2025-08-11T15:25:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T11:18:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201388124991697509943341813464340138962","date":"2025-08-05T01:50:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T07:00:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144569069857304252556963592129693153319","date":"2025-07-07T13:15:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-06T21:57:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-16T19:30:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-16T18:14:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Parkinson's Disease","date":"2025-06-13T20:15:49+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":"70cea121-8f5e-49ee-aa73-154fe1145adc","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51670628,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Parkinsons disease"},{"id":51670629,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Neurodegeneration"}],"tags":[],"updatedAt":"2026-02-16T16:00:39+00:00","versionOfRecord":{"articleIdentity":"rs-6890617","link":"https://doi.org/10.1038/s41531-026-01288-w","journal":{"identity":"npj-parkinsons-disease","isVorOnly":false,"title":"npj Parkinson's Disease"},"publishedOn":"2026-02-10 15:57:50","publishedOnDateReadable":"February 10th, 2026"},"versionCreatedAt":"2025-07-17 07:22:04","video":"","vorDoi":"10.1038/s41531-026-01288-w","vorDoiUrl":"https://doi.org/10.1038/s41531-026-01288-w","workflowStages":[]},"version":"v1","identity":"rs-6890617","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6890617","identity":"rs-6890617","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}