High molecular weight insoluble parkin in the substantia nigra of patients with idiopathic Parkinson’s disease

High molecular weight insoluble parkin in the substantia nigra of patients with idiopathic 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 High molecular weight insoluble parkin in the substantia nigra of patients with idiopathic Parkinson’s disease Cyntia Tremblay, Laura Pshevorskiy, Rosalie J. Cottez, Hélèna L. Denis, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8098616/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Parkinson’s disease (PD) is characterized by a loss of dopaminergic neurons and accumulation of α-synuclein (α-syn)-containing Lewy bodies in the substantia nigra (SN) pars compacta. Mutations in the gene coding for the protein parkin cause a form of autosomal recessive juvenile parkinsonism, but its role in idiopathic PD is poorly understood. Here, to investigate parkin changes in the SN in PD, we established a clinicopathology research platform comparing PD patients (n = 24) with Controls (n = 21). We first confirmed the massive loss of dopamine (DA) levels (-96%) in the putamen of PD patients, using HPLC/electrochemistry. Higher levels of phosphorylated α-syn (αsynP129) (23-fold) were observed in the SN of PD patients by Western immunoblotting. In formic acid extracts, an increase in the insoluble oligomeric form of parkin migrating at 260 kDa was observed (+ 49%) in the SN of PD patients, along with lower levels of the 55 kDa monomeric form (-47%). These changes in parkin were specific for the SN, and not observed in the putamen, parietal cortex and cerebellum. High molecular weight parkin correlated with αsynP129 levels and dopamine loss and was more prominently found in PD patients with levodopa-induced dyskinesias. Additional studies in animal models suggest that the aggregation of parkin is not a direct consequence of dopaminergic depletion or αsyn overproduction, but a component of PD cellular pathophysiology. Taken together, the results reported herein show that, beside dopamine loss and increased αsynP129, neurodegeneration in idiopathic PD is associated with a conversion of parkin into an insoluble high molecular weight form in the SN. Health sciences/Diseases Health sciences/Neurology Biological sciences/Neuroscience parkin PARK2 PRKN α-synuclein substantia nigra hyperphosphorylation dopamine putamen Parkinson’s Disease proteinopathy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder firstly characterized by the loss of dopaminergic neurons in the substantia nigra (SN) pars compacta (SNpc). This results in a massive decrease in the dopamine levels in the presynaptic terminals estimated to be between 60%-99% in caudate and putamen of patients [1‑4]. Restoring dopamine function in the basal ganglia remains the mainstay of clinical therapeutic interventions [5‑7]. In the last two decades, the PD research field has shifted its focus away from dopamine towards considering PD as an α-synuclein (αsyn) proteinopathy This view is chiefly based on genetic data and the observed presence of αsyn in Lewy bodies in the SN of PD patients [5, 8‑10]. The phosphorylation at serine 129 (αsynP129) is closely associated with abnormal αsyn aggregation [11‑14]. Duplication and triplication of the wild-type αsyn gene suffice to cause parkinsonism, suggesting that higher concentrations of the αsyn protein may be involved in sporadic PD [ 15 ]. From this came the hypothesis that simply reducing αsyn levels could be therapeutic [ 9 , 16 ], a view that has been recently disputed [17‑20]. Unfortunately, it must be recognized that, contrary to hypotheses based on dopamine loss, the focus on αsyn has not been particularly successful in yielding new therapeutic options for PD patients. Therefore, after more than 50 years following the discovery of dopamine loss in PD, the need to decipher new pathognomonic signs of PD within the SN remains dire. Genetic advances highlight the multifactorial and polygenic nature of PD [21‑23]. In particular, the loss of function of the ubiquitin E3 ligase parkin (aka PRKN) has been shown to cause genetic forms of PD [24‑26]. PRKN variants are the most common cause of autosomal recessive PD, accounting for > 40% of the familial early-onset cases [ 24 , 27 , 28 ]. Following activation by PINK1, parkin exerts a control on protein degradation, mitochondrial homeostasis and cellular mitophagy [ 24 , 29 , 30 ]. Parkin can undergo post-translational modifications (PTMs) such as phosphorylation, ubiquitination, sumoylation and neddylation as well nitrosylation, sulfhydration and sulfonation, which are thought to control parkin activity, its subcellular localization, conformation and solubility [ 24 , 31 ]. Decreased parkin solubility and formation of high-molecular-weight (HMW) parkin aggregates are reported with missense mutations or dopamine exposure, using in vitro experiments or in post-mortem tissues [32‑36]. More particularly, a recent study showed an association between a gradual increase in parkin oxidation and insolubility with age in multiple human brain regions, including the SN [ 37 ]. Such a loss of function of parkin could impair the ubiquitin-proteasome system, leading to oxidative damage and mitochondrial dysfunction, which is known in PD to contribute to degeneration of dopaminergic neurons [24, 38‑41]. In brief, while there is strong evidence for a role of parkin in various vital cell survival pathways, whether a parkin dysfunction contributes to idiopathic PD cases remains speculative. Relatively few clinicopathological studies have focused on the SN, despite the critical importance of this region in PD. Here, we took advantage of the Saskatchewan brain deposit on movement disorders providing detailed clinical patient characterization to investigate parkin and αsyn changes in terms of solubility and concentrations in multiple brain regions of idiopathic PD patients (n = 24) and controls individuals (n = 21), in relation with dopamine loss, disease duration and response to levodopa. This is the first demonstration that high molecular weight parkin aggregates accumulate in the SN of idiopathic PD patients. Results Higher post-mortem levels of insoluble high molecular weight (HMW) parkin in the SN of PD patients Despite the genetic link between parkin and PD, post-mortem parkin levels in the SN of idiopathic PD patients have not been investigated [ 42 ]. In SN homogenate fractions containing soluble proteins, we observed a band corresponding to parkin migrating at its expected molecular weight (55 kDa), with average levels being comparable between PD patients and controls (Fig. 1 a). Full images of Western blots (WB) are shown in Supplementary Fig. 1 . However, in fractions of insoluble proteins, beside the monomeric full-length parkin (55 kDa), we also detected an immunosignal at approximately 260 kDa, corresponding to a high molecular weight (HMW) form of parkin, akin to previous observations [ 32 , 35 , 37 , 43 ]. Interestingly, levels of this apparently aggregated form of parkin were higher in PD patients (+ 49%) along with lower levels of insoluble monomeric parkin (-47%) (Fig. 1 b-c), translating into a ratio of HMW/monomeric parkin that is twice higher in the SN of PD patients (Fig. 1 d). Adjustments made for age and sex did not change the statistical significance of the comparisons. Higher post-mortem levels of αsynP129 in the SN of PD patients Relatively few studies have documented post-mortem changes of αsyn in the SN of idiopathic PD patients and most have used qualitative immunofluorescence and immunohistochemistry [42, 44‑46]. Although αsynP129 is considered to be the most predominant αsyn PTM [11‑14, 20], few studies have sought to quantify it in the human SN. Therefore, we determined the levels of both αsynP129 and total αsyn in SN homogenates using WB. Full images of WB are shown in Supplementary Fig. 2 . In soluble fractions, the levels of αsynP129 were ~ 17 times higher in PD patients compared to controls (Fig. 2 a), whereas no significant differences were observed for total αsyn levels (Fig. 2 b,d). The ratio of αsynP129 over total αsyn (immunodetected using SYN1 or MJFR1 antibodies) was accordingly ~ 15 times higher in PD patients compared to controls (Fig. 2 c,e). In formic acid extracts containing detergent-insoluble proteins, average levels of αsynP129 were 22 times higher in PD patients compared to controls (Fig. 2 f). Total αsyn levels were slightly higher in PD, losing significance after adjustment for age and sex (Fig. 2 g,i). The corresponding αsyn phosphorylation ratio was ~ 18 or ~ 19 times higher in PD patients, respectively (Fig. 2 h,j). Adjustments made for age and sex did not change the strong statistical significance for comparisons of αsynP129 levels between groups. Changes in insoluble parkin in PD is restricted to the SN, whereas changes in phosphorylated α syn are more widespread Idiopathic PD is characterized by a remarkable vulnerability of dopaminergic neurons in the SNpc [ 47 , 48 ]. To investigate the regional specificity of parkin changes noted in the SN, similar experiments were performed in the putamen, cerebellum and parietal cortex. Full images of WB are shown in Supplementary Fig. 3 . In insoluble fractions from putamen, cerebellum and parietal cortex, both HMW and monomeric parkin could be detected. However, no significant differences between groups were observed (Fig. 3 a-f), except that both forms of parkin were less present in the PD group compared to the control group in the parietal cortex (Fig. 3 g-h). We also analyzed parkin immunosignal in tris-buffered saline (TBS) and detergent-soluble fractions from these three brain regions and no differences were found between groups ( Supplementary Figs. 4 & 6 along with full images of WB shown in Supplementary Figs. 5 & 7 ). Adjustments for age and sex did not change the statistical significance of these comparisons. Taken together, these data suggest that the conversion of monomeric parkin into an insoluble HMW form is a molecular outcome restricted to the SN in PD. Next, using the same experimental approach, we assessed changes in αsyn levels in the putamen, cerebellum and parietal cortex. In formic acid extracts, we first observed that the presence of αsynP129 was not detectable in these regions in a significant proportion of subjects, while total αsyn was more consistently present (Fig. 4 a-i). Full membrane images are depicted in Supplementary Fig. 8 . When comparing groups, despite large interindividual variability, we did observe statistically significant higher average levels of αsynP129 and higher αsynP129/total αsyn ratio in PD patients in cerebellum and parietal cortex (Fig. 4 d,f,g,i). After adjustments for age and sex, these differences remained significant only for the αsynP129/total αsyn ratio (Fig. 4 f,i). As for the total αsyn levels adjusted for age and sex, they were slightly lower in the parietal cortex (Fig. 4 h) in PD patients compared to the controls. In summary, despite a statistically significant higher phosphorylation status of αsyn in the cortex and cerebellum, the magnitude of the difference between idiopathic PD and controls was much more clear-cut in the SN than in other brain regions. Post-mortem levels of dopamine and metabolites in the putamen and SN of PD patients The loss of dopaminergic neurons in the SN is the main pathological hallmark of PD [1‑4]. Determining the concentrations of dopamine and its metabolites in the putamen remains the most quantitative assessment of nigrostriatal denervation. To that purpose, we utilized high-performance liquid chromatography with electrochemical detection (HPLC-EC). As anticipated, we found that PD patients display a massive reduction in the levels of dopamine (-96%) compared to controls (Fig. 5 a). Levels of homovanilic acid (HVA) (Fig. 5 b) and 3-methoxytyramine (3MT) (Fig. 5 c), which are metabolites of dopamine, were also reduced (-60% and − 89% versus Controls), but to a lesser extent than dopamine levels. Consequently, the metabolites/dopamine ratio was higher in PD patients by 10 times compared to controls (Fig. 5 d), consistent with an accelerated dopamine turnover in PD patients [1‑3]. A more commonly used but less quantitative index of nigrostriatal denervation, the tyrosine hydroxylase (TH) immunosignal was also reduced in both the SN and the putamen in PD patients compared to controls (-47% and − 82%, respectively) (Fig. 5 e,f). Full membrane images are shown in Supplementary Fig. 9 . Correlative analyses between parkin, αsyn, catecholamine and LRRK2 levels We then investigated the relationship between parkin and αsyn with nigral denervation. First, we observed a significant positive correlation between the HMW 260-kDa parkin and αsynP129 in insoluble fractions of the SN (Fig. 6 a). This association was also significant within PD patients (Fig. 6 b-c). By contrast, a weak inverse correlation was detected between monomeric parkin and αsynP129 in insoluble fractions of the SN (Fig. 6 a). Accordingly, the parkin HMW/monomers ratio was strongly associated with the accumulation of insoluble αsynP129 /total αsyn ratio (Fig. 6 c). No significant association was found for soluble parkin. Second, we observed an inverse association between the parkin aggregation ratio in the SN and dopamine in the putamen (Fig. 6 d). Weaker inverse correlations with TH levels of the SN were also noted (Fig. 6 d). The significance of the association between the parkin ratio and dopamine levels remained robust when analyzed specifically in PD patients (Fig. 6 e). Inverse correlations were also found between dopamine concentrations and soluble as well as insoluble αsynP129 (Fig. 6 d,f). No similar association between dopamine levels, αsyn and parkin levels were found in the putamen. Finally, given the importance of LRRK2 activity in PD [49‑51], we proceeded to the analysis of LRRK2 levels by ELISA in the SN and found strong correlations with insoluble 55-kDa parkin (inverse) and 260/55 parkin ratio (Fig. 6 g–h). These associations remained significant after adjustment for age and sex, indicating that parkin conversion into a HMW from may be related to elevated LRRK2 levels. In summary, the rise in parkin HMW species in the SN was associated with (i) αsynP129 and αsynP129/total αsyn, the latter only when including PD and controls, (ii) the extent of DAergic denervation, including within PD patients, and (iii) higher LRRK2 levels. Experimental nigral degeneration and α-synucleinopathy does not induce parkin aggregation Observations from post-mortem human PD samples do not conclusively reveal causal relationships, as these samples are collected significantly after the onset of pathological events. To get further insight into whether such an apparent aggregation of parkin is a consequence of dopaminergic neuronal loss, we measured parkin levels in mice and non-human primates exposed to the neurotoxin MPTP. Samples used here were from previous published studies in which MPTP non-human primates and mice had a 98% and 78% reduction of dopamine in the putamen, respectively [ 52 , 53 ]. We observed that the MPTP insult in non-human primates had no significant impact on soluble and insoluble monomeric parkin (55 kDa) or HMW parkin (Fig. 7 a-c). Contrasting with what we found in idiopathic PD, there was instead a trend toward higher monomeric parkin in MPTP-treated animals (Fig. 7 c). Five MPTP animals were treated with the mGlu5 receptor negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl) pyridine (MPEP) which was shown to reduce the development of levodopa-induced dyskinesias [ 54 ]. These animals displayed lower HMW parkin and 260/55 ratio (Fig. 7 b-d). A significant correlation between HMW parkin and dyskinesias score was observed (Fig. 7 g). No changes were detected in striata harvested from mice following the MPTP insult (Fig. 7 e). Full membrane images are shown in Supplementary Fig. 10–11 . This suggests that parkin aggregation cannot be replicated by simply generating dopaminergic neuronal loss. Finally, detergent-insoluble parkin was also investigated in cortices collected from an animal model of α-synucleinopathy (Thy1-αSyn mouse) [ 55 ], and no differences were found between groups (Fig. 7 f-h), suggesting that parkin aggregation is not a consequence of widespread synucleinopathy. Relationship between SN levels of HMW parkin and αsyn and clinical characteristics of the PD patients PD is a heterogenous disease with a wide clinical spectrum [ 8 , 56 , 57 ]. Taking advantage of the available clinical data, we first found no difference in insoluble parkin levels between PD patients with or without freezing of gait (FOG) (Fig. 8 a-c). When the levels of insoluble monomeric (55 kDa) parkin were examined in relation to disease duration, which lasted 13 years on average, a significant negative correlation was observed (Fig. 8 d). The parkin 260/55 kDa ratio positively correlated with disease duration, but only after adjusting for sex and age (Fig. 8 e). While parkin levels did not statistically differentiate PD patients with levodopa-induced complications (LIC) and those without, only PD subjects with LIC displayed a higher parkin 260/55 ratio than controls (Fig. 8 f-h). After separating according to the stages on Hoehn & Yahr Scale (H & Y), differences between parkin levels of the 2-3.5 score PD group and the 4–5 score PD group were nonsignificant (Fig. 8 i-k). By contrast, no significant association between SN levels of αsyn and FOG, disease duration, LIC and H & Y stages was detected (Fig. 8 l-v), suggesting that the increase in αsynP129 levels is a general characteristic of all patients with a PD diagnosis. Overall, these results indicate that the progression of PD is accompanied by a reduction in the monomeric form of parkin, which is converted into a HMW insoluble form, more prominently in individuals experiencing LIC. Discussion We investigated the relative concentrations and changes in solubility of parkin and αsyn in multiple brain regions of idiopathic PD patients (n = 24) followed in the Saskatchewan movement disorders program and control individuals (n = 21). In summary, in individuals with a PD diagnosis, we observed: (i) higher levels in the SN of insoluble HMW parkin migrating at 260 kDa, along with lower levels of 55-kDa monomers; (ii) higher levels of αsynP129 in the SN and other brain areas; (iii) a loss of dopamine in the putamen, which was inversely associated with HMW parkin and αsynP129 in the SN; and (iv) higher ratios of HMW/monomeric parkin in subjects with LIC and longer disease duration. This study adds parkin conversion into a HMW form in the SN as a novel pathognomonic sign of idiopathic PD, alongside with phosphorylation of α-synuclein at serine-129 in the SN and dopamine loss in the putamen. Post-transcriptional changes in parkin in the SN as a pathognomonic sign of idiopathic PD The main observation reported in the present manuscript is the higher post-mortem level of insoluble parkin migrating at 260 kDa combined with reduced monomers of parkin in the SN from PD patients. This observation suggests that a conversion of parkin into a HMW form occurs progressively in PD specifically in the SN. The concept of decreased parkin solubility has been previously suggested as a mechanism of loss of function following mutations [32‑37]. The parkin protein can undergo various post-transcriptional modifications (such as oxidation, S-nitrosylation, sulfonation and catecholation) affecting its structure and solubility, which could explain the HMW parkin species observed here [32, 35‑37, 43, 58, 59]. Three of these publications hinted toward higher insoluble monomeric parkin in the caudate/putamen in a smaller number sample from PD brains compared to controls, but levels in the SN were not examined [ 37 , 43 , 59 ]. A key post-mortem study reported that parkin solubility declined with age in the human brain, including the SN, in association with oxidative damage [ 37 ]. We observed no association with age, but this is likely due to the narrow range of ages in the individuals studied. More importantly, however, we did observe an association between a parkin conversion and longer disease duration, which suggests a key involvement of parkin in the PD neurodegeneration process. In sum, the present results offer the first evidence of the conversion of parkin into an insoluble HMW form as a pathophysiological event occurring in the SN of idiopathic PD patients. Possible role of a loss of function of parkin: relation with dopamine cell death Parkin conversion into an insoluble HMW form in the SN was associated with dopamine loss. Whether these pathological events occurred before, after or parallel to dopamine loss is unknown. However, the absence of significant changes in parkin following a massive nigrostriatal denervation in animals strongly suggests that the observed changes in parkin are not a mere consequence of dopamine loss. On the other hand, there is ample evidence in the literature that a loss of parkin function can affect dopamine cells. First, a loss of parkin activity is a key consequence of mutations in the gene coding for parkin, known to cause juvenile autosomal recessive PD [24‑26]. TH-positive neurons derived from induced pluripotent stem cells from PD patients with a parkin mutation also show defects in dopaminergic neurotransmission and toxicity [ 60 ]. A wealth of data from several experimental paradigms indicates that a loss of parkin enzymatic activity impairs proteasomal degradation of substrates, leading to defective mitochondria and oxidative damage, contributing to neurodegeneration [24, 38‑40, 61]. Given the association observed here between Parkin aggregation and higher LRRK2 levels, it becomes interesting to note that these two proteins are known to physically interact, and that enhanced LRRK2 activity was shown to reduce the mitophagic function of parkin in vitro [ 49 , 51 , 62 ]. Based on this, it could be hypothesized that the conversion of parkin into a HMW defective form could result in a loss of function contributing to the death of dopaminergic neurons in the SN. In sum, although the pathological role of sequestrating parkin into insoluble HMW forms remains to be elucidated, there are plausible mechanisms by which it can alter DAergic cell function and vulnerability in PD. Links between parkin and αsyn proteinopathies in PD For several decades, the αsyn protein has been one of the most extensively studied proteins in PD. This interest stems from its established role as a key component of Lewy bodies and genetic evidence linking mutations and gene triplications with parkinsonism[5, 8‑10, 15, 44‑46, 63]. Here, we found that relative levels of soluble and insoluble αsyn phosphorylated at serine 129 were dramatically increased in PD patients compared to controls in the SN as well as in other brain regions. By contrast, total αSyn levels in the SN, cerebellum, putamen, and cortex did not clearly distinguish Parkinson’s disease patients from controls. This implies that while formation of HMW parkin affects specifically the SN, the αsynP129-associated proteinopathy at least partly extends to other brain regions. These observations also suggest that therapeutic interventions simply aiming at decreasing the total amount of αsyn might not be sufficient without specifically targeting its hyperphosphorylation, as proposed previously [9, 16‑18, 64]. The accumulation of insoluble parkin species migrating at 260 kDa showed a significant association with levels of insoluble αSynP129 in the SN. This positive correlation may stem from impaired ubiquitin-proteasome system function, caused by reduced parkin activity, which would compromise the clearance of α-synuclein [ 30 , 32 , 65 ]. Notably, co-expression of parkin with α-synuclein in rats has been shown to promote PTMs of α-synuclein, particularly its phosphorylation at serine 129 [ 66 ]. Interestingly, our findings demonstrate that inducing synucleinopathy in mice does not lead to increased HMW parkin. This suggests that parkin dysfunction likely occurs in parallel or exert a causal role in the pathological mechanisms contributing to synucleinopathy and PD. Another notable observation was that HMW parkin rises as the disease progresses, whereas αsynP129 does not, indicating some divergence between the two pathological processes. In sum, the results of the present study, which analyzed both parkin and α-synuclein within the same sample series, point toward a possible link between these two proteinopathies and their specific detrimental effect on the dopaminergic system. Link between parkin and levodopa-induced complications Among PD patients, the ratio increase in HMW/monomeric parkin was significant only in those who developed motor complications following levodopa treatment (LIC). A possible explanation is that levodopa treatment could have triggered parkin aggregation in the SN, resulting in motor complications [ 67 , 68 ]. However, levodopa treatment of MPTP monkeys did induce dyskinesias, without leading to the formation of insoluble HMW parkin. Nevertheless, the anti-dyskinetic compound MPEP reduced the 260/55 ratio, suggesting that the absence of LIC in denervated animals is associated with a less conversion to HMW parkin. Several other common factors present in patients with LIC and parkin aggregates may also explain this association, including longer treatment and/or disease duration as well as more extensive DAergic denervation [ 69 , 70 ]. It remains interesting to consider that HMW parkin aggregates in PD patients could be a predictor for LIC. Limitations Although the described relative accumulation of parkin into a HMW species clearly distinguishes patients from controls, it is not well characterized. Additional techniques such as Fourier-transform infrared spectroscopy or circular dichroism spectroscopy would be necessary to assess secondary structure of parkin found in HMW forms, such as α-helical structures or those with β-pleated sheets [ 71 ]. Conclusions Overall, this study brings new insights on the implication of proteinopathies involving parkin and αsyn in idiopathic PD, possibly establishing parkin aggregation in the SN as a new pathognomonic marker for idiopathic PD, beyond α-synuclein phosphorylation at serine-129 and the loss of dopamine in the putamen. The association observed with the duration of the disease and the absence of similar changes in acute models of nigral denervation indicate that this modification of parkin is not a mere consequence, but an active player in the disease process. While the molecular characterization of HMW parkin remains to be performed, previous work shows that the expected loss of parkin function can contribute to mitochondrial dysfunction and nigral dopaminergic neurodegeneration. In addition to dopamine replacement and treatments targeting the reduction of α-synuclein phosphorylation at serine 129, blocking the conversion of parkin into its HMW form represents a potentially promising therapy for PD. Methodology Human Samples: Saskatchewan brain deposit on movement disorders Brain samples were obtained from the Saskatchewan Movement Disorders Program (SMDP) where clinical data collected from patients enabled longitudinal follow-up [ 56 , 72 ]. Consent for brain autopsy and use of brain tissue for research was approved by the hospital ethics committee and by the University of Saskatchewan Ethics Board. Two movement disorder neurologists performed all clinical diagnosis while post-mortem diagnosis was done by a certified neuropathologist. Data such as sex, age at onset, duration of disease, disease severity (Unified Parkinson’s Disease Rating Scale (UPDRS) and modified H & Y scale), prescribed drugs and their adverse effects (including motor complications) were collected. More specifically, disease duration (5–11 years: n = 11, 13–22 years: n = 13), motor complications (No-LIC: n = 7; LIC: n = 17) including dyskinesia and wearing off and were recorded during each assessment [ 56 , 73 ]. Freezing of gait (No-FOG: n = 8; FOG: n = 16) was also documented. The Hoehn & Yahr (H & Y) scale, being the most widely used and accepted staging system, was initially used in order to measure the global severity, followed by the use of the Unified Parkinson’s Disease Rating Scale (UPDRS) and modified H & Y scales (stages 2-3.5: n = 10; stages 4–5: n = 14) [ 56 , 73 , 74 ]. Since recruitment was done on a voluntary basis, the controls were slightly younger than the patients, while among the latter, the proportion of men was higher, similar to what is observed in the general population [ 75 ]. The cohort characteristics are shown in Supplementary Table 1 . Autopsy and Handling of the Brain Material After autopsies were performed within 24 hours after death, the collected brains were separated into two: one half of the brain, which was fixed in formalin, was used for histologic and diagnostic studies of the midbrain while the other half was frozen at -80°C and cut along the frontal plane in order to obtain 2–3 mm thick slices. Clinical evaluations were accomplished by one of two movement disorders neurologists (AHR, AR) and the post-mortem diagnosis was done for each patient by a Canadian certified neuropathologist [ 56 , 72 , 73 ]. Slices corresponding to the parietal cortex, the putamen and the SN (coronal plane) and the cerebellar cortex (axial plane) were used to extract tissues for these regions ( \(\:\sim\) 100 mg). Coronal slices containing the SN were cryostat-sectioned (20 µm), thaw-mounted onto SuperFrostPlus slides (75X50 mm), desiccated overnight at 4°C, and stored at -80°C until assayed, as described [ 76 ]. In addition, the SN were dissected on cryostat sections and 5 x 50 µm sections were harvested to obtain approximately 30 mg of frozen sample and stored at -80°C. Tissue pH was measured as an indication of tissue quality [ 76 , 77 ]. Tissue Processing For Western Blotting experiments, proteins were extracted from tissue homogenates by sequential fractionation using buffers, detergents and acid, as shown [ 76 , 78 , 79 ]. The TBS-soluble fraction contains mostly cytosolic and extracellular proteins, the detergent-soluble fraction includes most membrane-bound proteins and the detergent-insoluble fraction corresponds to insoluble proteins. Briefly, a homogenization in TBS buffer (50 mM tris-HCl, 138 mM NaCl, 2.7 mM KCl, with protease and phosphatase inhibitors and 0.1 mM EDTA) was first performed and samples were sonicated and centrifuged (20 min.; 100,000 g) to generate a supernatant, which is the TBS-soluble fraction. Secondly, the resulting pellet was subjected to homogenization in a lysis buffer containing detergents (0.5% of deoxycholate, 150 mM NaCl, 1% of Triton X-100, 10 mM NaH 2 PO 4 0.5% sodium dodecyl sulfate (SDS) with protease and phosphatase inhibitors and 0.1 mM EDTA), followed by sonication and centrifugation to generate a supernatant corresponding to the detergent-soluble fraction. Thirdly, the resulting pellet was resuspended in formic acid 99% (100 µl; Sigma-Adrich Cat# F0507), sonicated and centrifuged to produce the detergent-insoluble fraction (formic acid-soluble fraction containing insoluble proteins). The generated supernatant was then evaporated under a fume hood before solubilization in Laemmli buffer (60 mM Tris, 10% glycerol, 2% SDS, 0.0025% bromophenol blue, 2.5% β-mercaptoethanol, pH 8.5) and heated at 95°C for 5 minutes. Using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific), the protein quantity in the TBS-soluble and detergent-soluble fractions was determined. For the SN, since the available starting material was limited, only two fractionation steps were performed, resulting in a detergent-soluble fraction that also contained TBS-soluble proteins, and a detergent-insoluble fraction. Catecholamine Analysis Levels of dopamine, homovanillic acid (HVA) and 3-methoxytyramine (3MT) were determined by HPLC with electrochemical detection, as described previously [ 1 , 53 , 80 ]. Cold HClO 4 (0.1 N) was added to putamen sections, which were then homogenized and centrifuged for 10 minutes at 4°C (16,000g). Supernatants were collected and stored at -80°C while protein pellets were resuspended and icubated in TBS buffer 2X overnight at 4°C. The pellets were then vortexed, and the quantity of proteins was determined using the BCA protein assay kit (Thermo Fisher Scientific Cat #23225). Supernatants were subjected to HPLC coupled to electochemical detection (Waters, 717 plus Autosampler automatic injector, 1525 Binary Pump, 2465 Electrochemical Detector, Atlantis dC18 column). Standards of dopamine, HVA and 3-MT were injected in parallel to quantitate samples. LRRK2 ELISA For LRRK2 detection in SN detergent-soluble fraction, Meso Scale Discovery Technology was used (R-PLEX Human LRRK2 Assay Kit, # K1511PR, MSD, USA). Western Immunoblotting Proteins from TBS-soluble and detergent-soluble fractions were added to Laemmli 5X buffer and heated at 95°C for 5 minutes (denatured). The same amounts of proteins per sample (12 ug for SN and 15 µg for the remaining), were separated on a 14% sodium dodecyl-sulfate (SDS)-polyacrylamide gel by electrophoresis. The proteins were transferred on polyvinylidene fluoride (PVDF; Cytiva Life Sciences) 0.45 µm membranes. For membrane containing detergent insoluble fraction, total proteins were visualized with a No-Stain™ protein labeling reagent (Invitrogen by Thermo Fisher Scientific) before blocking to use as a loading control. For αsyn detection, the membranes were fixed with 4% paraformaldehyde pH 7.4 for 30 minutes before blocking. All membranes were blocked with 5% bovine serum albumin (BSA, BioShop Cat# ALB001) in phosphate-buffered saline (PBS)-Tween 0.1% (PBS, Fisher BioReagents Cat# BP399-20; Tween, Sigma-Aldrich) for 1h at ambient temperature. As for the immunoblots, the antibodies against parkin (Abcam Cat# ab77924 [PRK8], 1:1,000 & 1:1,300), TH (Pel-Freez Biologicals Cat# P40101-150, 1:1,000), αsyn (SYN1, BD Biosciences Cat# 610787, 1:1,000), MJFR1 (Abcam Cat# ab138501 [MJFR1], 1:1,000) and αsyn phosphorylated at serine 129 (αsynP129) (Abcam Cat# ab168381 [MJF-R13 (8–8)], 1:1,000; Abcam Cat# ab51253 [EP1536Y] 1:500) were used. As a loading control for the TBS-soluble ant detergent-soluble fractions, the antibody against β-actin (Applied Biological Materials Cat# G043) was used at 1:5,000. All incubations were done in Superblock™ blocking buffer in PBS (Thermo Fisher Scientific) containing 0.1% Tween 20 and 0,05% sodium azide. After incubation with a primary antibody, the membranes were washed in PBS-Tween 0.1%, followed by an incubation with a horseradish peroxidase (HRP) anti-mouse (Jackson ImmunoResearch Labs) or anti-rabbit secondary antibody (Jackson ImmunoResearch Labs) at 1:40,000 in PBS containing 0.1% Tween 20 and 1% BSA. The detections were done with Amersham Imager 680 (GE Healthcare Bio-Sciences) following revelation with Luminata (Sigma-Aldrich Millipore), a chemiluminescence HRP substrate. For the analysis of band intensity, the Image Lab software (Bio-Rad) was used. Immunoblots are shown in Supplementary Fig. 1–3, 5, 7–9 & 11–13 . Animals: MPTP monkeys Drug-naive ovariectomized female cynomolgus monkeys ( macaca fascicularis ) were continuously injected with MPTP in order to induce a stable parkinsonian syndrome, which was followed by a levodopa/benserazide treatment while four intact monkeys were used as controls, as previously described [ 52 , 54 ]. The SN was dissected from frozen section as detailed elsewhere [ 52 ]. The detergent-soluble and the detergent-insoluble fractions were obtained following the method described above. Animals: αsyn mice Male Thy1-αsyn (transgenic, Tg; n = 48) and nontransgenic C57BL/6 (NonTg; n = 28–31) mice were bred in our animal research facility from Thy1.2-αsyn mice (line 61) on a full C57BL/6 background, as described previously [ 55 ]. Briefly, mice were subjected to a 12:12h dark:light cycle and were kept in ventilated cages, where one contained between 2 to 5 mice, in addition of having free access to water and fed with different diets (formulated control, no DHA or enriched DHA) not relevant for the present study [ 55 ]. Only male mice were used for experimentations because of the transgene’s location on chromosome X and all protocols were approved by the animal research committee of the Centre de recherche du CHU de Québec-Université Lava [ 55 ]. Animals: MPTP mice Male C57BL/6 mice were injected with a MPTP \(\:\bullet\:\) HCl solution (7 i.p injections) freshly dissolved in 0.9% saline at 4 months of age, where the MPTP administration was performed twice on the first two days of the experimental protocol at 12h-intervals, and once a day on the three subsequent days while the remaining mice were injected with 0.9% saline i.p, as previously described [ 53 ]. Briefly, mice were kept in groups of 3 to 4 per cage and had free access to food and water. As for the MPTP doses, they were calculated for each mouse with respect to the body surface area. Statistical Analysis To compare two groups, Mann-Whitney tests were used except in some instances unpaired student’s t-test were used when normal distribution and equal variance were assumed. For comparisons involved two or more groups, the parametric one-way ANOVA test as well as the nonparametric Kruskall-Wallis test depending on the normality test result were done. Following the one-way ANOVA test, the Tukey’s multiple comparisons test was done as a post-hoc test while the Kruskall-Wallis test was followed by the Dunn’s multiple comparisons test. Correlative analyses were performed using Pearson or Spearman correlation tests, depending on data distribution. Data were adjusted for age at death and/or sex using distribution and multivariate analyses. Normalization in the detergent-insoluble fractions were done over the weight of the appropriate brain region (mg) while in the other analyzed fractions, the normalization was done over β-actin. A statistical comparison corresponds to a p-value lower than 0.05. All statistical analyses were done using GraphPad Prism 9.0 or JMP 16.2.0 software. Data availability The data supporting findings of the present study are available from the corresponding author on a reasonable request. Abbreviations asyn, a-synuclein; asynP129, asyn phosphorylated at serine 129; 3MT, 3-methoxytyramine; BCA, bicinchoninic acid; BSA, bovide serum albumin; DA, dopamine; DAPI, 4’,6-diamidino-2-phenylindole; H&Y, Hoehn & Yahr HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; HVA, homovanillic acid; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine PBS, phosphate-buffered saline; PD, Parkinson’s Disease; prk, parkin; PTMs, post-translational modifications; PVDF, polyvinylidene fluoride; RT, room temperature; SDS, sodium dodecyl-sulfate; SN, Substantia nigra; TBS, tris-buffered saline; TH, tyrosine hydroxylase; UV, ultraviolet; WB, western blots; Declarations Data availability The data supporting findings of the present study are available from the corresponding author on a reasonable request. Acknowledgements All authors and particularly A. R. and A.H.R., are grateful for unrestricted research support from the Dr. Ali Rajput Endowment for Parkinson’s Disease and Movement Disorders. Funding Declaration This work was supported by grants from Parkinson Canada to F.C and A.J.R (2017-1110), the Canadian Institutes of Health Research (CIHR) to F.C. [grant numbers PJT 168927] and Canada foundation for innovation to F.C (#34480). R.J.C. holds a Master’s scholarship from the « Fond d’enseignement et de recherche du cercle du congrès de la Faculté de pharmacie, Université Laval». F.C. was a Fonds de recherche du Québec-Sante (FRQ-S) research scholar. Author Contributions L.P, C.T, H.D and V.E performed experiments and analyzed data. C.T accomplished human tissue processing. R.J.C. contributed to the revision of the manuscript. M.M and T.D.P provided the animal samples (MPTP monkeys). Clinical assessment of patients was done by A.H.R. and A.R. Both A.H.R. and F.C designed the study. L.P and F.C wrote the manuscript. 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14:34:48","extension":"html","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":183149,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/d3ca845418cadb2f616c58b8.html"},{"id":97266562,"identity":"f9984f53-0bd5-4cb5-8bbc-7382a4e10ca7","added_by":"auto","created_at":"2025-12-02 14:34:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":251567,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigher HMW and lower monomers of insoluble parkin in the substantia nigra of PD patients.\u003c/strong\u003e While detergent-soluble native parkin (55 kDa) levels were unchanged (\u003cstrong\u003ea\u003c/strong\u003e), higher levels of detergent-insoluble HMW parkin (260 kDa) (\u003cstrong\u003eb\u003c/strong\u003e) and lower levels of detergent-insoluble native parkin (55 kDa) (\u003cstrong\u003ec\u003c/strong\u003e) were detected in the SN of PD patients compared to controls. This leads to a higher detergent-insoluble parkin (260/55) ratio in the SN of the PD group compared to the control group (\u003cstrong\u003ed\u003c/strong\u003e). Statistical analysis: data are represented as mean ± SEM (N= 21 ctrl and 24 PD); P-value from Mann-Whitney tests are shown on top, while P-value after adjustments for age and sex are provided below the graphs. Data in b and c were normalized with SN sample weight (mg).\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003eAbbreviations: Ctrl/C, control individuals; HMW, high molecular weight; PD/P, Parkinson’s disease patients; SEM, standard error of the mean; SN, substantia nigra. O.D., Optical Density. \u003c/em\u003eRepresentative WB of consecutive bands were shown from the samples. Full images are shown in Supplementary Fig. 1.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/e46c142b10aec8b8015c64c4.png"},{"id":97368502,"identity":"b69597ef-4464-4b21-ad0d-09c751bf10d1","added_by":"auto","created_at":"2025-12-03 16:22:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":356706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigher post-mortem levels of asynP129 in the substantia nigra of PD patients.\u003c/strong\u003e Levels of asynP129 were higher in detergent-soluble and detergent-insoluble fractions extracted from the SN of PD patients (a, f). Differences in total asyn levels when measured with SYN1 and MJFR1 antibody were overall nonsignificant between groups in detergent-soluble (b, d) detergent-insoluble fractions (g, i). Accordingly, the asynP129/total asyn ratios were also higher in PD in both fractions (c, e, h j). Statistical analysis: data are represented as mean ± SEM (N= 21 ctrl and 24 PD); P-value from Mann-Whitney tests are shown on top, while P-value after adjustments for age and sex are provided below the graphs. The normalization of O.D. values in the detergent-insoluble fraction was done over the SN weight (mg). \u003cem\u003eAbbreviations: Ctrl/C, control individuals; PD/P, Parkinson’s disease patients; SEM, standard error of the mean; SN, substantia nigra; asyn, a-synuclein; asynP129, a-synuclein phosphorylated at serine 129; O.D., Optical Density\u003c/em\u003e. Representative WB of consecutive bands were shown from the samples. Full images are shown in Supplementary Fig. 2.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/259d5e6c739b3440b8dcd08c.png"},{"id":97266564,"identity":"a740e92e-7d43-41be-8642-5848f8606355","added_by":"auto","created_at":"2025-12-02 14:34:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":445419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePost-mortem levels of detergent-insoluble parkin in putamen, cerebellum and parietal cortex of PD patients. \u003c/strong\u003eNo differences were detected between controls and PD patients for parkin aggregates (260 kDa), native parkin (55 kDa) and the parkin ratio (260/55) both in the putamen (\u003cstrong\u003ea-c\u003c/strong\u003e) and the cerebellum (\u003cstrong\u003ed-f\u003c/strong\u003e). In the parietal cortex, although PD patients had lower levels of parkin HMW species (\u003cstrong\u003eg\u003c/strong\u003e) and parkin native proteins (\u003cstrong\u003eh\u003c/strong\u003e), the differences in the parkin ratio levels remained nonsignificant (\u003cstrong\u003ei\u003c/strong\u003e). Statistical analysis: data are represented as mean ± SEM (Putamen, N= 9 ctrl and 22 PD; Cerebellum, N= 22 ctrl and 16 PD; Parietal Cortex, N= 16 ctrl and 9 PD); unpaired Student T test (\u003cstrong\u003ea, g, i\u003c/strong\u003e); Mann-Whitney test (\u003cstrong\u003eb-f, h\u003c/strong\u003e); the P-value after adjustments for age and sex is provided; All O.D. values from detergent-insoluble fractions were normalized over sample weight (mg). \u003cem\u003eAbbreviations: Ctrl/C, control individuals; PD/P, Parkinson’s disease patients; HMW, high molecular weight; SEM, standard error of the mean; O.D., Optical Density. \u003c/em\u003eRepresentative WB of bands were shown from the samples, where the inserted black vertical line indicates nonconsecutive bands. Full imagesare shown in Supplementary Fig. 3.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/fa6c19266e19c1f8b3a59d0f.png"},{"id":97266571,"identity":"7af0dc86-b46a-4e64-a351-54106d96e8e3","added_by":"auto","created_at":"2025-12-02 14:34:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":395046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePost-mortem levels of detergent-insoluble αsyn in putamen, cerebellum and parietal cortex of PD patients. \u003c/strong\u003eIn putamen, cerebellum and parietal cortex, PD patients had higher levels of asynP129 \u003cstrong\u003e(a, d, g)\u003c/strong\u003e and asynP129/total asyn ratio (\u003cstrong\u003ec, f, i), \u003c/strong\u003ewith or withoutadjustments for sex and age (except in the cortex where significance of asynP129 was lost after adjustment). By contrast, no significant differences in total asyn (\u003cstrong\u003eb, e, h\u003c/strong\u003e) levels were noted between groups in these regions. Statistical analysis: data are represented as mean ± SEM (Putamen, N= 9 ctrl and 22 PD; Cerebellum, N= 22 ctrl and 16 PD; Parietal Cortex, N= 16 ctrl and 9 PD); P values shown are from Mann-Whitney (top) and analysis of covariance with age and sex as covariates (bottom); All O.D. values from detergent-insoluble fractions were normalized over sample weight (mg). \u003cem\u003eAbbreviations: Ctrl/C, controls; PD/P, Parkinson’s disease patients; asyn, a-synuclein; asynP129, a-synuclein phosphorylated at serine 129; SEM, standard error of the mean; O.D., Optical Density. \u003c/em\u003eRepresentative WB of bands are shown, where the inserted black vertical line indicates nonconsecutive bands. Full images are in Supplementary Fig. 8.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/30d6b69fa57e53cbe67c7a4a.png"},{"id":97368501,"identity":"0423234c-bbb5-4799-acc7-ffc6053c5072","added_by":"auto","created_at":"2025-12-03 16:22:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":358804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePost-mortem levels of dopamine, metabolites HVA and 3MT and TH in the putamen and in the SN of PD patients. (a)\u003c/strong\u003e DA concentrations were decreased by over 95% in the putamen of PD patients compared to controls. (\u003cstrong\u003eb,c)\u003c/strong\u003e Levels of HVA and 3MT, metabolites of DA, were reduced in the putamen of patients with PD, along with a higher (HVA+3MT)/DA ratio (\u003cstrong\u003ed\u003c/strong\u003e). TH immunosignal normalized to b-actin in the SN (\u003cstrong\u003ee\u003c/strong\u003e) and putamen (\u003cstrong\u003ef\u003c/strong\u003e) were lower in PD patients compared to controls. Statistical analysis: data are represented as mean ± SEM (Putamen, N= 9 ctrl and 22 PD; Substantia Nigra, N= 21 ctrl and 23 PD); Mann-Whitney test (\u003cstrong\u003ea-d\u003c/strong\u003e); unpaired student T test (\u003cstrong\u003ee-f\u003c/strong\u003e); the P-value after adjustments for age and sex is provided. \u003cem\u003eAbbreviations: Ctrl/C, control individuals; PD/P, Parkinson’s disease patients; SN, substantia nigra; DA, dopamine; HVA, homovanillic acid; 3MT, 3-methoxytyramine; TH, tyrosine hydroxylase; SEM, standard error of the mean;\u003c/em\u003e \u003cem\u003eO.D., Optical Density.\u003c/em\u003e Representative WB of consecutive bands were shown from the samples. Full images are shown in Supplementary Figure 9.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/0f3d68314176aca27f44d780.png"},{"id":97266574,"identity":"cc1f3dd5-ad3b-46fb-919e-a7380bb04aed","added_by":"auto","created_at":"2025-12-02 14:34:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":409035,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epost-mortem\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e parkin, asyn in the SN and dopamine or TH in the putamen, and LRRK2 in the SN of PD patients. (a)\u003c/strong\u003e The correlations of asyn levels with parkin levels were computed in the SN. Positive correlations between the αsynP129 levels and parkin 260 kDa (\u003cstrong\u003eb\u003c/strong\u003e) and between the αsynP129/Total αsyn and parkin ratio (\u003cstrong\u003ec\u003c/strong\u003e) are shown. Correlations of putamen levels TH and DA with SN levels of parkin and αsyn were also analyzed (\u003cstrong\u003ed\u003c/strong\u003e). Negative correlations between DA levels in the putamen and the SN parkin ratio and (\u003cstrong\u003ee\u003c/strong\u003e) with the SN ratio of αsynP129/Total αsyn (\u003cstrong\u003ef\u003c/strong\u003e) are shown. LRRK2 SN levels correlate negatively with soluble parkin (55 kDa) (\u003cstrong\u003eg\u003c/strong\u003e) and positively with the insoluble Parkin ratio (\u003cstrong\u003eh\u003c/strong\u003e). Statistical analysis: Spearman’s rank correlations were conducted to evaluate associations among the studied variables. Simple linear regressions were performed to generate coefficients of determination (r²). Correlations were adjusted for age and sex to generate p-values (•p \u0026lt; 0.05; ••p \u0026lt; 0.01; •••p \u0026lt; 0.001; ••••p \u0026lt; 0.0001). Adjusted p-values are provided for correlations in the full cohort as well as for correlations calculated within the PD group only. Red and blue cells indicate significant positive and negative correlations, respectively. Red rectangles around cells in (\u003cstrong\u003ea\u003c/strong\u003e) and (\u003cstrong\u003ed\u003c/strong\u003e) correspond to the selected correlations shown in (\u003cstrong\u003eb–c\u003c/strong\u003e) and (\u003cstrong\u003ee–f\u003c/strong\u003e), respectively. Panels (\u003cstrong\u003eb, c, e, f, g, h\u003c/strong\u003e) underwent the same analyses: linear regression for r², Pearson or Spearman correlation depending on normality, and adjusted correlations for age*sex in the full cohort and in the PD group. \u003cem\u003eAbbreviations: Ctrl/C, control individuals; PD/P, Parkinson’s disease patients; SN, substantia nigra; asyn, a-synuclein; asynP129, a-synuclein phosphorylated at serine 129; DA, dopamine; TH, tyrosine hydroxylase; O.D., Optical Density.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/8d89095d4cd682d77487c36e.png"},{"id":97368268,"identity":"4b2d22a5-4b77-48c1-b847-4ed66247e832","added_by":"auto","created_at":"2025-12-03 16:21:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":570131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePost-mortem levels of parkin in SN of MPTP-treated non-human primates, in striatum of MPTP mice and in cortex of α-synuclein mice. \u003c/strong\u003eNative monomeric detergent-soluble parkin (55 kDa) levels appeared higher in the MPTP + L-DOPA group compared to the intact group, without reaching statistical significance (\u003cstrong\u003ea\u003c/strong\u003e) although in the detergent-soluble fraction it the 55 KDa parkin level are non-significantly lower in the intact group (\u003cstrong\u003ec\u003c/strong\u003e). MPEP reduced parkin aggregates (260 kDa) in comparison with the MPTP+L-Dopa treated group while the parkin ratio (260/55) decreased compared to the intact group (\u003cstrong\u003eb\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e). (\u003cstrong\u003ee\u003c/strong\u003e). Positive correlation between detergent-insoluble Parkin 260 KDa and dyskinesia score. In MPTP-denervated mice, the differences in native parkin levels were nonsignificant (\u003cstrong\u003ef\u003c/strong\u003e) in the detergent-soluble fraction of the striatum In αsyn transgenic mice,\u003cstrong\u003e \u003c/strong\u003eno differences were detected between the NonTg and Tg (αsyn) groups for parkin aggregates (260 kDa), monomeric parkin (55 kDa) and the parkin ratio (260/55) levels in the detergent-insoluble fraction of the cortex (\u003cstrong\u003eg-i\u003c/strong\u003e). Statistical analysis: data are represented as mean ± SEM (\u003cu\u003eMPTP monkeys: N= 4 intact group, 4 MPTP group, 5 MPTP+L-DOPA group, 5 MPTP+L-DOPA+MPEP group\u003c/u\u003e; αsyn mice: N= 28-31 NonTg and 48 Tg (αsyn); MPTP mice: N= 8 saline and 6 MPTP); \u003cu\u003eKruskal-Wallis test followed by Dunn’s multiple comparisons test (\u003c/u\u003e\u003cu\u003e\u003cstrong\u003ea-d\u003c/strong\u003e\u003c/u\u003e\u003cu\u003e); \u003c/u\u003eunpaired student T test (\u003cstrong\u003ef-g\u003c/strong\u003e); Mann-Whitney test (\u003cstrong\u003eh-i\u003c/strong\u003e); the normalization of O.D. values in the detergent-soluble fraction was done over β-actin while the normalization in the detergent-insoluble fraction was done over mg of total protein (primates) and Nostain values (mice). Legend for MPTP monkeys WB: 1, intact; 2, MPTP; 3, MPTP+DOPA; 4, MPTP+L-DOPA+MPEP. Legend for αsyn mice WB: 1, NonTg; 2, Tg (αsyn). Legend for MPTP mice WB: 1, saline; 2, MPTP. \u003cem\u003eAbbreviations: MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; DOPA, levodopa; MPEP, 2-methyl-6; SN, substantia nigra; αsyn, α-synuclein; NonTg, nontransgenic; Tg, transgenic; SEM, standard error of the mean; O.D., Optical Density. \u003c/em\u003eRepresentative WB of bands were shown from the samples, where the inserted black vertical line indicates nonconsecutive bands. Full imagesare shown in Supplementary Fig. 11-13.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/d2cff60bc0f583bf49088111.png"},{"id":97367899,"identity":"34c3f77c-10a9-48a1-b75e-a7e8cde8a5cb","added_by":"auto","created_at":"2025-12-03 16:20:59","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":833834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetergent-insoluble levels of parkin and asyn in SN in relation to clinical data. \u003c/strong\u003eDetergent-insoluble parkin (260 kDa), native proteins (55 kDa) and parkin ratio (260/55) are shown in relation to FOG (\u003cstrong\u003ea-c\u003c/strong\u003e), to LIC (\u003cstrong\u003eg-i\u003c/strong\u003e) and to H \u0026amp; Y (\u003cstrong\u003ej-l\u003c/strong\u003e). The correlations of parkin and parkin ratio levels of PD patients with disease duration were computed (\u003cstrong\u003ed-e\u003c/strong\u003e). Briefly, when the PD patients were separated into two groups according to the presence of FOG, the presence of LIC or the stages on the H \u0026amp; Y scale, no differences were observed between the two PD groups, except for the LIC patients that had higher parkin ratio levels. In addition, parkin ratio levels positively correlated with disease duration, after adjusting for sex and age.\u003cstrong\u003e \u003c/strong\u003eDetergent-insoluble asynP129, total asyn and the asynP129 over the total asyn ratio are shown in relation to FOG (\u003cstrong\u003el-n\u003c/strong\u003e), to LIC (\u003cstrong\u003eq-s\u003c/strong\u003e) and to H \u0026amp; Y higher (\u003cstrong\u003et-v\u003c/strong\u003e). The correlations of asynP129 and asyn ratio levels of PD patients with disease duration were also computed (\u003cstrong\u003eo-p\u003c/strong\u003e). Statistical analysis: data are represented as mean ± SEM (N= 21 ctrl and 24 PD); one-way ANOVA followed by Tukey’s or Dunnett’s post-hoc tests (\u003cstrong\u003ea, f, i, m, r, u\u003c/strong\u003e); Kruskal-Wallis test followed by Dunn’s multiple comparisons test (\u003cstrong\u003eb-c, g-h, j-l, n, q, s-t, v\u003c/strong\u003e); * p \u0026lt;0.05; simple linear regressions were performed to generate coefficients of determination (r\u003csup\u003e2\u003c/sup\u003e); correlations were adjusted for age and sex to generate p-values. The adjusted p-value according to sex and age is provided; the normalization of O.D. values in the detergent-insoluble fraction was done over the SN weight (mg). \u003cem\u003eAbbreviations: Ctrl, control individuals; PD, Parkinson’s disease patients; SN, substantia nigra; asyn, a-synuclein; asynP129, a-synuclein phosphorylated at serine 129; O.D., Optical Density; FOG, freezing of gait; LIC, levodopa-induced complications; H \u0026amp; Y, Hoehn \u0026amp; Yahr Scale; SEM, standard error of the mean; ns, nonsignificant.\u003c/em\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/e390a47cd7a88344272bdb4a.png"},{"id":97372748,"identity":"6c37a84b-624e-4275-8359-67e90ac31a58","added_by":"auto","created_at":"2025-12-03 16:33:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5111053,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/22f90e8d-94e4-4cf6-827c-14d163ce45b2.pdf"},{"id":97266577,"identity":"01c85cc7-29f1-4ac4-a9c4-f826642ecd34","added_by":"auto","created_at":"2025-12-02 14:34:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":8543283,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryParkinasynNPJoct2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-8098616/v1/d295d82ecbb9cd35d34ddc2e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High molecular weight insoluble parkin in the substantia nigra of patients with idiopathic Parkinson’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a progressive neurodegenerative disorder firstly characterized by the loss of dopaminergic neurons in the substantia nigra (SN) pars compacta (SNpc). This results in a massive decrease in the dopamine levels in the presynaptic terminals estimated to be between 60%-99% in caudate and putamen of patients [1‑4]. Restoring dopamine function in the basal ganglia remains the mainstay of clinical therapeutic interventions [5‑7]. In the last two decades, the PD research field has shifted its focus away from dopamine towards considering PD as an α-synuclein (αsyn) proteinopathy This view is chiefly based on genetic data and the observed presence of αsyn in Lewy bodies in the SN of PD patients [5, 8‑10]. The phosphorylation at serine 129 (αsynP129) is closely associated with abnormal αsyn aggregation [11‑14]. Duplication and triplication of the wild-type αsyn gene suffice to cause parkinsonism, suggesting that higher concentrations of the αsyn protein may be involved in sporadic PD [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. From this came the hypothesis that simply reducing αsyn levels could be therapeutic [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], a view that has been recently disputed [17‑20]. Unfortunately, it must be recognized that, contrary to hypotheses based on dopamine loss, the focus on αsyn has not been particularly successful in yielding new therapeutic options for PD patients. Therefore, after more than 50 years following the discovery of dopamine loss in PD, the need to decipher new pathognomonic signs of PD within the SN remains dire.\u003c/p\u003e\u003cp\u003eGenetic advances highlight the multifactorial and polygenic nature of PD [21‑23]. In particular, the loss of function of the ubiquitin E3 ligase parkin (aka PRKN) has been shown to cause genetic forms of PD [24‑26]. PRKN variants are the most common cause of autosomal recessive PD, accounting for \u0026gt;\u0026thinsp;40% of the familial early-onset cases [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Following activation by PINK1, parkin exerts a control on protein degradation, mitochondrial homeostasis and cellular mitophagy [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Parkin can undergo post-translational modifications (PTMs) such as phosphorylation, ubiquitination, sumoylation and neddylation as well nitrosylation, sulfhydration and sulfonation, which are thought to control parkin activity, its subcellular localization, conformation and solubility [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Decreased parkin solubility and formation of high-molecular-weight (HMW) parkin aggregates are reported with missense mutations or dopamine exposure, using \u003cem\u003ein vitro\u003c/em\u003e experiments or in \u003cem\u003epost-mortem\u003c/em\u003e tissues [32‑36]. More particularly, a recent study showed an association between a gradual increase in parkin oxidation and insolubility with age in multiple human brain regions, including the SN [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Such a loss of function of parkin could impair the ubiquitin-proteasome system, leading to oxidative damage and mitochondrial dysfunction, which is known in PD to contribute to degeneration of dopaminergic neurons [24, 38‑41]. In brief, while there is strong evidence for a role of parkin in various vital cell survival pathways, whether a parkin dysfunction contributes to idiopathic PD cases remains speculative.\u003c/p\u003e\u003cp\u003eRelatively few clinicopathological studies have focused on the SN, despite the critical importance of this region in PD. Here, we took advantage of the Saskatchewan brain deposit on movement disorders providing detailed clinical patient characterization to investigate parkin and αsyn changes in terms of solubility and concentrations in multiple brain regions of idiopathic PD patients (n\u0026thinsp;=\u0026thinsp;24) and controls individuals (n\u0026thinsp;=\u0026thinsp;21), in relation with dopamine loss, disease duration and response to levodopa. This is the first demonstration that high molecular weight parkin aggregates accumulate in the SN of idiopathic PD patients.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eHigher post-mortem levels of insoluble high molecular weight (HMW) parkin in the SN of PD patients\u003c/em\u003e\u003c/p\u003e\u003cp\u003eDespite the genetic link between parkin and PD, \u003cem\u003epost-mortem\u003c/em\u003e parkin levels in the SN of idiopathic PD patients have not been investigated [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In SN homogenate fractions containing soluble proteins, we observed a band corresponding to parkin migrating at its expected molecular weight (55 kDa), with average levels being comparable between PD patients and controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Full images of Western blots (WB) are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e. However, in fractions of insoluble proteins, beside the monomeric full-length parkin (55 kDa), we also detected an immunosignal at approximately 260 kDa, corresponding to a high molecular weight (HMW) form of parkin, akin to previous observations [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Interestingly, levels of this apparently aggregated form of parkin were higher in PD patients (+\u0026thinsp;49%) along with lower levels of insoluble monomeric parkin (-47%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-c), translating into a ratio of HMW/monomeric parkin that is twice higher in the SN of PD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Adjustments made for age and sex did not change the statistical significance of the comparisons.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eHigher post-mortem levels of αsynP129 in the SN of PD patients\u003c/h2\u003e\u003cp\u003eRelatively few studies have documented \u003cem\u003epost-mortem\u003c/em\u003e changes of αsyn in the SN of idiopathic PD patients and most have used qualitative immunofluorescence and immunohistochemistry [42, 44‑46]. Although αsynP129 is considered to be the most predominant αsyn PTM [11‑14, 20], few studies have sought to quantify it in the human SN. Therefore, we determined the levels of both αsynP129 and total αsyn in SN homogenates using WB. Full images of WB are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e. In soluble fractions, the levels of αsynP129 were ~\u0026thinsp;17 times higher in PD patients compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), whereas no significant differences were observed for total αsyn levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,d). The ratio of αsynP129 over total αsyn (immunodetected using SYN1 or MJFR1 antibodies) was accordingly\u0026thinsp;~\u0026thinsp;15 times higher in PD patients compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,e). In formic acid extracts containing detergent-insoluble proteins, average levels of αsynP129 were 22 times higher in PD patients compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Total αsyn levels were slightly higher in PD, losing significance after adjustment for age and sex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg,i). The corresponding αsyn phosphorylation ratio was ~\u0026thinsp;18 or ~\u0026thinsp;19 times higher in PD patients, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh,j). Adjustments made for age and sex did not change the strong statistical significance for comparisons of αsynP129 levels between groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eChanges in insoluble parkin in PD is restricted to the SN, whereas changes in phosphorylated\u003c/em\u003e α\u003cem\u003esyn are more widespread\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIdiopathic PD is characterized by a remarkable vulnerability of dopaminergic neurons in the SNpc [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. To investigate the regional specificity of parkin changes noted in the SN, similar experiments were performed in the putamen, cerebellum and parietal cortex. Full images of WB are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e. In insoluble fractions from putamen, cerebellum and parietal cortex, both HMW and monomeric parkin could be detected. However, no significant differences between groups were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-f), except that both forms of parkin were less present in the PD group compared to the control group in the parietal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-h). We also analyzed parkin immunosignal in tris-buffered saline (TBS) and detergent-soluble fractions from these three brain regions and no differences were found between groups (\u003cb\u003eSupplementary Figs.\u0026nbsp;4 \u0026amp; 6\u003c/b\u003e along with full images of WB shown in \u003cb\u003eSupplementary Figs.\u0026nbsp;5 \u0026amp; 7\u003c/b\u003e). Adjustments for age and sex did not change the statistical significance of these comparisons. Taken together, these data suggest that the conversion of monomeric parkin into an insoluble HMW form is a molecular outcome restricted to the SN in PD.\u003c/p\u003e\u003cp\u003eNext, using the same experimental approach, we assessed changes in αsyn levels in the putamen, cerebellum and parietal cortex. In formic acid extracts, we first observed that the presence of αsynP129 was not detectable in these regions in a significant proportion of subjects, while total αsyn was more consistently present (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-i). Full membrane images are depicted in \u003cb\u003eSupplementary Fig.\u0026nbsp;8\u003c/b\u003e. When comparing groups, despite large interindividual variability, we did observe statistically significant higher average levels of αsynP129 and higher αsynP129/total αsyn ratio in PD patients in cerebellum and parietal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed,f,g,i). After adjustments for age and sex, these differences remained significant only for the αsynP129/total αsyn ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef,i). As for the total αsyn levels adjusted for age and sex, they were slightly lower in the parietal cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh) in PD patients compared to the controls. In summary, despite a statistically significant higher phosphorylation status of αsyn in the cortex and cerebellum, the magnitude of the difference between idiopathic PD and controls was much more clear-cut in the SN than in other brain regions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePost-mortem levels of dopamine and metabolites in the putamen and SN of PD patients\u003c/h3\u003e\n\u003cp\u003eThe loss of dopaminergic neurons in the SN is the main pathological hallmark of PD [1‑4]. Determining the concentrations of dopamine and its metabolites in the putamen remains the most quantitative assessment of nigrostriatal denervation. To that purpose, we utilized high-performance liquid chromatography with electrochemical detection (HPLC-EC). As anticipated, we found that PD patients display a massive reduction in the levels of dopamine (-96%) compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Levels of homovanilic acid (HVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) and 3-methoxytyramine (3MT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), which are metabolites of dopamine, were also reduced (-60% and \u0026minus;\u0026thinsp;89% versus Controls), but to a lesser extent than dopamine levels. Consequently, the metabolites/dopamine ratio was higher in PD patients by 10 times compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed), consistent with an accelerated dopamine turnover in PD patients [1‑3]. A more commonly used but less quantitative index of nigrostriatal denervation, the tyrosine hydroxylase (TH) immunosignal was also reduced in both the SN and the putamen in PD patients compared to controls (-47% and \u0026minus;\u0026thinsp;82%, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee,f). Full membrane images are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;9\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCorrelative analyses between parkin, αsyn, catecholamine and LRRK2 levels\u003c/h3\u003e\n\u003cp\u003eWe then investigated the relationship between parkin and αsyn with nigral denervation. First, we observed a significant positive correlation between the HMW 260-kDa parkin and αsynP129 in insoluble fractions of the SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). This association was also significant within PD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-c). By contrast, a weak inverse correlation was detected between monomeric parkin and αsynP129 in insoluble fractions of the SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Accordingly, the parkin HMW/monomers ratio was strongly associated with the accumulation of insoluble αsynP129 /total αsyn ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). No significant association was found for soluble parkin. Second, we observed an inverse association between the parkin aggregation ratio in the SN and dopamine in the putamen (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Weaker inverse correlations with TH levels of the SN were also noted (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The significance of the association between the parkin ratio and dopamine levels remained robust when analyzed specifically in PD patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Inverse correlations were also found between dopamine concentrations and soluble as well as insoluble αsynP129 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed,f). No similar association between dopamine levels, αsyn and parkin levels were found in the putamen. Finally, given the importance of LRRK2 activity in PD [49‑51], we proceeded to the analysis of LRRK2 levels by ELISA in the SN and found strong correlations with insoluble 55-kDa parkin (inverse) and 260/55 parkin ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg\u0026ndash;h). These associations remained significant after adjustment for age and sex, indicating that parkin conversion into a HMW from may be related to elevated LRRK2 levels. In summary, the rise in parkin HMW species in the SN was associated with (i) αsynP129 and αsynP129/total αsyn, the latter only when including PD and controls, (ii) the extent of DAergic denervation, including within PD patients, and (iii) higher LRRK2 levels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eExperimental nigral degeneration and α-synucleinopathy does not induce parkin aggregation\u003c/h3\u003e\n\u003cp\u003eObservations from \u003cem\u003epost-mortem\u003c/em\u003e human PD samples do not conclusively reveal causal relationships, as these samples are collected significantly after the onset of pathological events. To get further insight into whether such an apparent aggregation of parkin is a consequence of dopaminergic neuronal loss, we measured parkin levels in mice and non-human primates exposed to the neurotoxin MPTP. Samples used here were from previous published studies in which MPTP non-human primates and mice had a 98% and 78% reduction of dopamine in the putamen, respectively [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We observed that the MPTP insult in non-human primates had no significant impact on soluble and insoluble monomeric parkin (55 kDa) or HMW parkin (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-c). Contrasting with what we found in idiopathic PD, there was instead a trend toward higher monomeric parkin in MPTP-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Five MPTP animals were treated with the mGlu5 receptor negative allosteric modulator (NAM) 2-methyl-6-(phenylethynyl) pyridine (MPEP) which was shown to reduce the development of levodopa-induced dyskinesias [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These animals displayed lower HMW parkin and 260/55 ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-d). A significant correlation between HMW parkin and dyskinesias score was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eg). No changes were detected in striata harvested from mice following the MPTP insult (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Full membrane images are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;10\u0026ndash;11\u003c/b\u003e. This suggests that parkin aggregation cannot be replicated by simply generating dopaminergic neuronal loss.\u003c/p\u003e\u003cp\u003eFinally, detergent-insoluble parkin was also investigated in cortices collected from an animal model of α-synucleinopathy (Thy1-αSyn mouse) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and no differences were found between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef-h), suggesting that parkin aggregation is not a consequence of widespread synucleinopathy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eRelationship between SN levels of HMW parkin and αsyn and clinical characteristics of the PD patients\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePD is a heterogenous disease with a wide clinical spectrum [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Taking advantage of the available clinical data, we first found no difference in insoluble parkin levels between PD patients with or without freezing of gait (FOG) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea-c). When the levels of insoluble monomeric (55 kDa) parkin were examined in relation to disease duration, which lasted 13 years on average, a significant negative correlation was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). The parkin 260/55 kDa ratio positively correlated with disease duration, but only after adjusting for sex and age (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). While parkin levels did not statistically differentiate PD patients with levodopa-induced complications (LIC) and those without, only PD subjects with LIC displayed a higher parkin 260/55 ratio than controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef-h). After separating according to the stages on Hoehn \u0026amp; Yahr Scale (H \u0026amp; Y), differences between parkin levels of the 2-3.5 score PD group and the 4\u0026ndash;5 score PD group were nonsignificant (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ei-k). By contrast, no significant association between SN levels of αsyn and FOG, disease duration, LIC and H \u0026amp; Y stages was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003el-v), suggesting that the increase in αsynP129 levels is a general characteristic of all patients with a PD diagnosis. Overall, these results indicate that the progression of PD is accompanied by a reduction in the monomeric form of parkin, which is converted into a HMW insoluble form, more prominently in individuals experiencing LIC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e We investigated the relative concentrations and changes in solubility of parkin and αsyn in multiple brain regions of idiopathic PD patients (n\u0026thinsp;=\u0026thinsp;24) followed in the Saskatchewan movement disorders program and control individuals (n\u0026thinsp;=\u0026thinsp;21). In summary, in individuals with a PD diagnosis, we observed: (i) higher levels in the SN of insoluble HMW parkin migrating at 260 kDa, along with lower levels of 55-kDa monomers; (ii) higher levels of αsynP129 in the SN and other brain areas; (iii) a loss of dopamine in the putamen, which was inversely associated with HMW parkin and αsynP129 in the SN; and (iv) higher ratios of HMW/monomeric parkin in subjects with LIC and longer disease duration. This study adds parkin conversion into a HMW form in the SN as a novel pathognomonic sign of idiopathic PD, alongside with phosphorylation of α-synuclein at serine-129 in the SN and dopamine loss in the putamen.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePost-transcriptional changes in parkin in the SN as a pathognomonic sign of idiopathic PD\u003c/h2\u003e\u003cp\u003eThe main observation reported in the present manuscript is the higher \u003cem\u003epost-mortem\u003c/em\u003e level of insoluble parkin migrating at 260 kDa combined with reduced monomers of parkin in the SN from PD patients. This observation suggests that a conversion of parkin into a HMW form occurs progressively in PD specifically in the SN. The concept of decreased parkin solubility has been previously suggested as a mechanism of loss of function following mutations [32‑37]. The parkin protein can undergo various post-transcriptional modifications (such as oxidation, S-nitrosylation, sulfonation and catecholation) affecting its structure and solubility, which could explain the HMW parkin species observed here [32, 35‑37, 43, 58, 59]. Three of these publications hinted toward higher insoluble monomeric parkin in the caudate/putamen in a smaller number sample from PD brains compared to controls, but levels in the SN were not examined [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. A key \u003cem\u003epost-mortem\u003c/em\u003e study reported that parkin solubility declined with age in the human brain, including the SN, in association with oxidative damage [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We observed no association with age, but this is likely due to the narrow range of ages in the individuals studied. More importantly, however, we did observe an association between a parkin conversion and longer disease duration, which suggests a key involvement of parkin in the PD neurodegeneration process. In sum, the present results offer the first evidence of the conversion of parkin into an insoluble HMW form as a pathophysiological event occurring in the SN of idiopathic PD patients.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePossible role of a loss of function of parkin: relation with dopamine cell death\u003c/h3\u003e\n\u003cp\u003eParkin conversion into an insoluble HMW form in the SN was associated with dopamine loss. Whether these pathological events occurred before, after or parallel to dopamine loss is unknown. However, the absence of significant changes in parkin following a massive nigrostriatal denervation in animals strongly suggests that the observed changes in parkin are not a mere consequence of dopamine loss. On the other hand, there is ample evidence in the literature that a loss of parkin function can affect dopamine cells. First, a loss of parkin activity is a key consequence of mutations in the gene coding for parkin, known to cause juvenile autosomal recessive PD [24‑26]. TH-positive neurons derived from induced pluripotent stem cells from PD patients with a parkin mutation also show defects in dopaminergic neurotransmission and toxicity [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. A wealth of data from several experimental paradigms indicates that a loss of parkin enzymatic activity impairs proteasomal degradation of substrates, leading to defective mitochondria and oxidative damage, contributing to neurodegeneration [24, 38‑40, 61]. Given the association observed here between Parkin aggregation and higher LRRK2 levels, it becomes interesting to note that these two proteins are known to physically interact, and that enhanced LRRK2 activity was shown to reduce the mitophagic function of parkin in vitro [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Based on this, it could be hypothesized that the conversion of parkin into a HMW defective form could result in a loss of function contributing to the death of dopaminergic neurons in the SN. In sum, although the pathological role of sequestrating parkin into insoluble HMW forms remains to be elucidated, there are plausible mechanisms by which it can alter DAergic cell function and vulnerability in PD.\u003c/p\u003e\n\u003ch3\u003eLinks between parkin and αsyn proteinopathies in PD\u003c/h3\u003e\n\u003cp\u003eFor several decades, the αsyn protein has been one of the most extensively studied proteins in PD. This interest stems from its established role as a key component of Lewy bodies and genetic evidence linking mutations and gene triplications with parkinsonism[5, 8‑10, 15, 44‑46, 63]. Here, we found that relative levels of soluble and insoluble αsyn phosphorylated at serine 129 were dramatically increased in PD patients compared to controls in the SN as well as in other brain regions. By contrast, total αSyn levels in the SN, cerebellum, putamen, and cortex did not clearly distinguish Parkinson\u0026rsquo;s disease patients from controls. This implies that while formation of HMW parkin affects specifically the SN, the αsynP129-associated proteinopathy at least partly extends to other brain regions. These observations also suggest that therapeutic interventions simply aiming at decreasing the total amount of αsyn might not be sufficient without specifically targeting its hyperphosphorylation, as proposed previously [9, 16‑18, 64].\u003c/p\u003e\u003cp\u003eThe accumulation of insoluble parkin species migrating at 260 kDa showed a significant association with levels of insoluble αSynP129 in the SN. This positive correlation may stem from impaired ubiquitin-proteasome system function, caused by reduced parkin activity, which would compromise the clearance of α-synuclein [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Notably, co-expression of parkin with α-synuclein in rats has been shown to promote PTMs of α-synuclein, particularly its phosphorylation at serine 129 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Interestingly, our findings demonstrate that inducing synucleinopathy in mice does not lead to increased HMW parkin. This suggests that parkin dysfunction likely occurs in parallel or exert a causal role in the pathological mechanisms contributing to synucleinopathy and PD. Another notable observation was that HMW parkin rises as the disease progresses, whereas αsynP129 does not, indicating some divergence between the two pathological processes. In sum, the results of the present study, which analyzed both parkin and α-synuclein within the same sample series, point toward a possible link between these two proteinopathies and their specific detrimental effect on the dopaminergic system.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLink between parkin and levodopa-induced complications\u003c/h2\u003e\u003cp\u003eAmong PD patients, the ratio increase in HMW/monomeric parkin was significant only in those who developed motor complications following levodopa treatment (LIC). A possible explanation is that levodopa treatment could have triggered parkin aggregation in the SN, resulting in motor complications [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. However, levodopa treatment of MPTP monkeys did induce dyskinesias, without leading to the formation of insoluble HMW parkin. Nevertheless, the anti-dyskinetic compound MPEP reduced the 260/55 ratio, suggesting that the absence of LIC in denervated animals is associated with a less conversion to HMW parkin. Several other common factors present in patients with LIC and parkin aggregates may also explain this association, including longer treatment and/or disease duration as well as more extensive DAergic denervation [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. It remains interesting to consider that HMW parkin aggregates in PD patients could be a predictor for LIC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eAlthough the described relative accumulation of parkin into a HMW species clearly distinguishes patients from controls, it is not well characterized. Additional techniques such as Fourier-transform infrared spectroscopy or circular dichroism spectroscopy would be necessary to assess secondary structure of parkin found in HMW forms, such as α-helical structures or those with β-pleated sheets [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOverall, this study brings new insights on the implication of proteinopathies involving parkin and αsyn in idiopathic PD, possibly establishing parkin aggregation in the SN as a new pathognomonic marker for idiopathic PD, beyond α-synuclein phosphorylation at serine-129 and the loss of dopamine in the putamen. The association observed with the duration of the disease and the absence of similar changes in acute models of nigral denervation indicate that this modification of parkin is not a mere consequence, but an active player in the disease process. While the molecular characterization of HMW parkin remains to be performed, previous work shows that the expected loss of parkin function can contribute to mitochondrial dysfunction and nigral dopaminergic neurodegeneration. In addition to dopamine replacement and treatments targeting the reduction of α-synuclein phosphorylation at serine 129, blocking the conversion of parkin into its HMW form represents a potentially promising therapy for PD.\u003c/p\u003e"},{"header":"Methodology","content":"\u003ch2\u003eHuman Samples: Saskatchewan brain deposit on movement disorders\u003c/h2\u003e\u003cp\u003eBrain samples were obtained from the Saskatchewan Movement Disorders Program (SMDP) where clinical data collected from patients enabled longitudinal follow-up [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Consent for brain autopsy and use of brain tissue for research was approved by the hospital ethics committee and by the University of Saskatchewan Ethics Board. Two movement disorder neurologists performed all clinical diagnosis while post-mortem diagnosis was done by a certified neuropathologist. Data such as sex, age at onset, duration of disease, disease severity (Unified Parkinson’s Disease Rating Scale (UPDRS) and modified H \u0026amp; Y scale), prescribed drugs and their adverse effects (including motor complications) were collected. More specifically, disease duration (5–11 years: n = 11, 13–22 years: n = 13), motor complications (No-LIC: n = 7; LIC: n = 17) including dyskinesia and wearing off and were recorded during each assessment [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Freezing of gait (No-FOG: n = 8; FOG: n = 16) was also documented. The Hoehn \u0026amp; Yahr (H \u0026amp; Y) scale, being the most widely used and accepted staging system, was initially used in order to measure the global severity, followed by the use of the Unified Parkinson’s Disease Rating Scale (UPDRS) and modified H \u0026amp; Y scales (stages 2-3.5: n = 10; stages 4–5: n = 14) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Since recruitment was done on a voluntary basis, the controls were slightly younger than the patients, while among the latter, the proportion of men was higher, similar to what is observed in the general population [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The cohort characteristics are shown in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e\u003ch2\u003eAutopsy and Handling of the Brain Material\u003c/h2\u003e\u003cp\u003eAfter autopsies were performed within 24 hours after death, the collected brains were separated into two: one half of the brain, which was fixed in formalin, was used for histologic and diagnostic studies of the midbrain while the other half was frozen at -80°C and cut along the frontal plane in order to obtain 2–3 mm thick slices. Clinical evaluations were accomplished by one of two movement disorders neurologists (AHR, AR) and the post-mortem diagnosis was done for each patient by a Canadian certified neuropathologist [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Slices corresponding to the parietal cortex, the putamen and the SN (coronal plane) and the cerebellar cortex (axial plane) were used to extract tissues for these regions (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\sim\\)\u003c/span\u003e\u003c/span\u003e100 mg). Coronal slices containing the SN were cryostat-sectioned (20 µm), thaw-mounted onto SuperFrostPlus slides (75X50 mm), desiccated overnight at 4°C, and stored at -80°C until assayed, as described [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. In addition, the SN were dissected on cryostat sections and 5 x 50 µm sections were harvested to obtain approximately 30 mg of frozen sample and stored at -80°C. Tissue pH was measured as an indication of tissue quality [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eTissue Processing\u003c/h2\u003e\u003cp\u003eFor Western Blotting experiments, proteins were extracted from tissue homogenates by sequential fractionation using buffers, detergents and acid, as shown [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The TBS-soluble fraction contains mostly cytosolic and extracellular proteins, the detergent-soluble fraction includes most membrane-bound proteins and the detergent-insoluble fraction corresponds to insoluble proteins. Briefly, a homogenization in TBS buffer (50 mM tris-HCl, 138 mM NaCl, 2.7 mM KCl, with protease and phosphatase inhibitors and 0.1 mM EDTA) was first performed and samples were sonicated and centrifuged (20 min.; 100,000 g) to generate a supernatant, which is the TBS-soluble fraction. Secondly, the resulting pellet was subjected to homogenization in a lysis buffer containing detergents (0.5% of deoxycholate, 150 mM NaCl, 1% of Triton X-100, 10 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.5% sodium dodecyl sulfate (SDS) with protease and phosphatase inhibitors and 0.1 mM EDTA), followed by sonication and centrifugation to generate a supernatant corresponding to the detergent-soluble fraction. Thirdly, the resulting pellet was resuspended in formic acid 99% (100 µl; Sigma-Adrich Cat# F0507), sonicated and centrifuged to produce the detergent-insoluble fraction (formic acid-soluble fraction containing insoluble proteins). The generated supernatant was then evaporated under a fume hood before solubilization in Laemmli buffer (60 mM Tris, 10% glycerol, 2% SDS, 0.0025% bromophenol blue, 2.5% β-mercaptoethanol, pH 8.5) and heated at 95°C for 5 minutes. Using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific), the protein quantity in the TBS-soluble and detergent-soluble fractions was determined. For the SN, since the available starting material was limited, only two fractionation steps were performed, resulting in a detergent-soluble fraction that also contained TBS-soluble proteins, and a detergent-insoluble fraction.\u003c/p\u003e\u003ch2\u003eCatecholamine Analysis\u003c/h2\u003e\u003cp\u003eLevels of dopamine, homovanillic acid (HVA) and 3-methoxytyramine (3MT) were determined by HPLC with electrochemical detection, as described previously [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Cold HClO\u003csub\u003e4\u003c/sub\u003e (0.1 N) was added to putamen sections, which were then homogenized and centrifuged for 10 minutes at 4°C (16,000g). Supernatants were collected and stored at -80°C while protein pellets were resuspended and icubated in TBS buffer 2X overnight at 4°C. The pellets were then vortexed, and the quantity of proteins was determined using the BCA protein assay kit (Thermo Fisher Scientific Cat #23225). Supernatants were subjected to HPLC coupled to electochemical detection (Waters, 717 plus Autosampler automatic injector, 1525 Binary Pump, 2465 Electrochemical Detector, Atlantis dC18 column). Standards of dopamine, HVA and 3-MT were injected in parallel to quantitate samples.\u003c/p\u003e\u003ch2\u003eLRRK2 ELISA\u003c/h2\u003e\u003cp\u003eFor LRRK2 detection in SN detergent-soluble fraction, Meso Scale Discovery Technology was used (R-PLEX Human LRRK2 Assay Kit, # K1511PR, MSD, USA).\u003c/p\u003e\u003ch2\u003eWestern Immunoblotting\u003c/h2\u003e\u003cp\u003eProteins from TBS-soluble and detergent-soluble fractions were added to Laemmli 5X buffer and heated at 95°C for 5 minutes (denatured). The same amounts of proteins per sample (12 ug for SN and 15 µg for the remaining), were separated on a 14% sodium dodecyl-sulfate (SDS)-polyacrylamide gel by electrophoresis. The proteins were transferred on polyvinylidene fluoride (PVDF; Cytiva Life Sciences) 0.45 µm membranes. For membrane containing detergent insoluble fraction, total proteins were visualized with a No-Stain™ protein labeling reagent (Invitrogen by Thermo Fisher Scientific) before blocking to use as a loading control. For αsyn detection, the membranes were fixed with 4% paraformaldehyde pH 7.4 for 30 minutes before blocking. All membranes were blocked with 5% bovine serum albumin (BSA, BioShop Cat# ALB001) in phosphate-buffered saline (PBS)-Tween 0.1% (PBS, Fisher BioReagents Cat# BP399-20; Tween, Sigma-Aldrich) for 1h at ambient temperature. As for the immunoblots, the antibodies against parkin (Abcam Cat# ab77924 [PRK8], 1:1,000 \u0026amp; 1:1,300), TH (Pel-Freez Biologicals Cat# P40101-150, 1:1,000), αsyn (SYN1, BD Biosciences Cat# 610787, 1:1,000), MJFR1 (Abcam Cat# ab138501 [MJFR1], 1:1,000) and αsyn phosphorylated at serine 129 (αsynP129) (Abcam Cat# ab168381 [MJF-R13 (8–8)], 1:1,000; Abcam Cat# ab51253 [EP1536Y] 1:500) were used. As a loading control for the TBS-soluble ant detergent-soluble fractions, the antibody against β-actin (Applied Biological Materials Cat# G043) was used at 1:5,000. All incubations were done in Superblock™ blocking buffer in PBS (Thermo Fisher Scientific) containing 0.1% Tween 20 and 0,05% sodium azide. After incubation with a primary antibody, the membranes were washed in PBS-Tween 0.1%, followed by an incubation with a horseradish peroxidase (HRP) anti-mouse (Jackson ImmunoResearch Labs) or anti-rabbit secondary antibody (Jackson ImmunoResearch Labs) at 1:40,000 in PBS containing 0.1% Tween 20 and 1% BSA. The detections were done with Amersham Imager 680 (GE Healthcare Bio-Sciences) following revelation with Luminata (Sigma-Aldrich Millipore), a chemiluminescence HRP substrate. For the analysis of band intensity, the Image Lab software (Bio-Rad) was used. Immunoblots are shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;1–3, 5, 7–9 \u0026amp; 11–13\u003c/b\u003e.\u003c/p\u003e\u003ch2\u003eAnimals: MPTP monkeys\u003c/h2\u003e\u003cp\u003eDrug-naive ovariectomized female cynomolgus monkeys (\u003cem\u003emacaca fascicularis\u003c/em\u003e) were continuously injected with MPTP in order to induce a stable parkinsonian syndrome, which was followed by a levodopa/benserazide treatment while four intact monkeys were used as controls, as previously described [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The SN was dissected from frozen section as detailed elsewhere [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The detergent-soluble and the detergent-insoluble fractions were obtained following the method described above.\u003c/p\u003e\u003ch2\u003eAnimals: αsyn mice\u003c/h2\u003e\u003cp\u003eMale Thy1-αsyn (transgenic, Tg; n = 48) and nontransgenic C57BL/6 (NonTg; n = 28–31) mice were bred in our animal research facility from Thy1.2-αsyn mice (line 61) on a full C57BL/6 background, as described previously [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Briefly, mice were subjected to a 12:12h dark:light cycle and were kept in ventilated cages, where one contained between 2 to 5 mice, in addition of having free access to water and fed with different diets (formulated control, no DHA or enriched DHA) not relevant for the present study [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Only male mice were used for experimentations because of the transgene’s location on chromosome X and all protocols were approved by the animal research committee of the Centre de recherche du CHU de Québec-Université Lava [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eAnimals: MPTP mice\u003c/h2\u003e\u003cp\u003eMale C57BL/6 mice were injected with a MPTP\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\bullet\\:\\)\u003c/span\u003e\u003c/span\u003eHCl solution (7 i.p injections) freshly dissolved in 0.9% saline at 4 months of age, where the MPTP administration was performed twice on the first two days of the experimental protocol at 12h-intervals, and once a day on the three subsequent days while the remaining mice were injected with 0.9% saline i.p, as previously described [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Briefly, mice were kept in groups of 3 to 4 per cage and had free access to food and water. As for the MPTP doses, they were calculated for each mouse with respect to the body surface area.\u003c/p\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eTo compare two groups, Mann-Whitney tests were used except in some instances unpaired student’s t-test were used when normal distribution and equal variance were assumed. For comparisons involved two or more groups, the parametric one-way ANOVA test as well as the nonparametric Kruskall-Wallis test depending on the normality test result were done. Following the one-way ANOVA test, the Tukey’s multiple comparisons test was done as a post-hoc test while the Kruskall-Wallis test was followed by the Dunn’s multiple comparisons test. Correlative analyses were performed using Pearson or Spearman correlation tests, depending on data distribution. Data were adjusted for age at death and/or sex using distribution and multivariate analyses. Normalization in the detergent-insoluble fractions were done over the weight of the appropriate brain region (mg) while in the other analyzed fractions, the normalization was done over β-actin. A statistical comparison corresponds to a p-value lower than 0.05. All statistical analyses were done using GraphPad Prism 9.0 or JMP 16.2.0 software.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eThe data supporting findings of the present study are available from the corresponding author on a reasonable request.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003easyn,\u0026nbsp;a-synuclein;\u003c/p\u003e\n\u003cp\u003easynP129,\u0026nbsp;asyn phosphorylated at serine 129;\u003c/p\u003e\n\u003cp\u003e3MT, 3-methoxytyramine;\u003c/p\u003e\n\u003cp\u003eBCA, bicinchoninic acid;\u003c/p\u003e\n\u003cp\u003eBSA, bovide serum albumin;\u003c/p\u003e\n\u003cp\u003eDA, dopamine;\u003c/p\u003e\n\u003cp\u003eDAPI, 4\u0026rsquo;,6-diamidino-2-phenylindole;\u003c/p\u003e\n\u003cp\u003eH\u0026amp;Y, Hoehn \u0026amp; Yahr\u003c/p\u003e\n\u003cp\u003eHPLC, high-performance liquid chromatography;\u003c/p\u003e\n\u003cp\u003eHRP, horseradish peroxidase;\u003c/p\u003e\n\u003cp\u003eHVA, homovanillic acid;\u003c/p\u003e\n\u003cp\u003eMPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine\u003c/p\u003e\n\u003cp\u003ePBS, phosphate-buffered saline;\u003c/p\u003e\n\u003cp\u003ePD, Parkinson\u0026rsquo;s Disease;\u003c/p\u003e\n\u003cp\u003eprk, parkin;\u003c/p\u003e\n\u003cp\u003ePTMs, post-translational modifications;\u003c/p\u003e\n\u003cp\u003ePVDF, polyvinylidene fluoride;\u003c/p\u003e\n\u003cp\u003eRT, room temperature;\u003c/p\u003e\n\u003cp\u003eSDS, sodium dodecyl-sulfate;\u003c/p\u003e\n\u003cp\u003eSN, Substantia nigra;\u003c/p\u003e\n\u003cp\u003eTBS, tris-buffered saline;\u003c/p\u003e\n\u003cp\u003eTH, tyrosine hydroxylase;\u003c/p\u003e\n\u003cp\u003eUV, ultraviolet;\u003c/p\u003e\n\u003cp\u003eWB, western blots;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting findings of the present study are available from the corresponding author on a reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors and particularly A. R. and A.H.R., are grateful for unrestricted research support from the Dr. Ali Rajput Endowment for Parkinson\u0026rsquo;s Disease and Movement Disorders.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from Parkinson Canada to F.C and A.J.R (2017-1110), the Canadian Institutes of Health Research (CIHR) to F.C. [grant numbers PJT 168927] and Canada foundation for innovation to F.C (#34480). R.J.C. holds a Master\u0026rsquo;s scholarship from the \u0026laquo; Fond d\u0026rsquo;enseignement et de recherche du cercle du congr\u0026egrave;s de la Facult\u0026eacute; de pharmacie, Universit\u0026eacute; Laval\u0026raquo;. F.C. was a Fonds de recherche du Qu\u0026eacute;bec-Sante (FRQ-S) research scholar.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eL.P, C.T, H.D and V.E performed experiments and analyzed data. C.T accomplished human tissue processing. R.J.C. contributed to the revision of the manuscript. M.M and T.D.P provided the animal samples (MPTP monkeys). Clinical assessment of patients was done by A.H.R. and A.R. Both A.H.R. and F.C designed the study. L.P and F.C wrote the manuscript. All authors were involved in the final submission and revisions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no competing interests.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCalon F, Morissette M, Rajput AH, Hornykiewicz O, Bedard PJ, Di Paolo T. Changes of GABA receptors and dopamine turnover in the postmortem brains of parkinsonians with levodopa-induced motor complications. Mov Disord. 2003;18(3):241-253.\u003c/li\u003e\n\u003cli\u003eWilson JM et al. 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Neural Regen Res. 2025;20(2):574-586.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-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":"parkin, PARK2, PRKN, α-synuclein, substantia nigra, hyperphosphorylation, dopamine, putamen, Parkinson’s Disease, proteinopathy","lastPublishedDoi":"10.21203/rs.3.rs-8098616/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8098616/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is characterized by a loss of dopaminergic neurons and accumulation of α-synuclein (α-syn)-containing Lewy bodies in the substantia nigra (SN) pars compacta. Mutations in the gene coding for the protein parkin cause a form of autosomal recessive juvenile parkinsonism, but its role in idiopathic PD is poorly understood. Here, to investigate parkin changes in the SN in PD, we established a clinicopathology research platform comparing PD patients (n\u0026thinsp;=\u0026thinsp;24) with Controls (n\u0026thinsp;=\u0026thinsp;21). We first confirmed the massive loss of dopamine (DA) levels (-96%) in the putamen of PD patients, using HPLC/electrochemistry. Higher levels of phosphorylated α-syn (αsynP129) (23-fold) were observed in the SN of PD patients by Western immunoblotting. In formic acid extracts, an increase in the insoluble oligomeric form of parkin migrating at 260 kDa was observed (+\u0026thinsp;49%) in the SN of PD patients, along with lower levels of the 55 kDa monomeric form (-47%). These changes in parkin were specific for the SN, and not observed in the putamen, parietal cortex and cerebellum. High molecular weight parkin correlated with αsynP129 levels and dopamine loss and was more prominently found in PD patients with levodopa-induced dyskinesias. Additional studies in animal models suggest that the aggregation of parkin is not a direct consequence of dopaminergic depletion or αsyn overproduction, but a component of PD cellular pathophysiology. Taken together, the results reported herein show that, beside dopamine loss and increased αsynP129, neurodegeneration in idiopathic PD is associated with a conversion of parkin into an insoluble high molecular weight form in the SN.\u003c/p\u003e","manuscriptTitle":"High molecular weight insoluble parkin in the substantia nigra of patients with idiopathic Parkinson’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-02 14:34:43","doi":"10.21203/rs.3.rs-8098616/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T12:48:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T20:43:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-01T11:01:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119882490447367707222001536749309298062","date":"2025-11-30T11:57:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144569069857304252556963592129693153319","date":"2025-11-28T16:32:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"115472598745714005374525182247984194304","date":"2025-11-28T13:48:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"278834630089343554460834321519609855231","date":"2025-11-28T12:18:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-28T11:50:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-17T13:55:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-17T10:38:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Parkinson's Disease","date":"2025-11-12T17:02:35+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":"ee40efe1-6041-4eb3-90a7-d8ac48e41dc3","owner":[],"postedDate":"December 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":58842565,"name":"Health sciences/Diseases"},{"id":58842566,"name":"Health sciences/Neurology"},{"id":58842567,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-04-20T12:10:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-02 14:34:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8098616","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8098616","identity":"rs-8098616","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}