Amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotides targeting α-synuclein as a novel therapy for Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. A characteristic pathological feature of PD is cytoplasmic accumulation of α-synuclein (SNCA) protein. Multiplication of the SNCA gene in familial PD and pathological accumulation of SNCA protein during progression of sporadic PD suggest that increased SNCA protein levels increase the risk of PD. Thus, reducing SNCA expression levels could delay PD onset or modify the disease course. For efficient knock down, we designed and synthesized an amido-bridged nucleic acids (AmNA)-modified antisense oligonucleotide (ASO) that targeted SNCA with improved stability and cellular uptake in vivo. AmNA-ASO efficiently downregulated SNCA at both the mRNA and protein level in vitro and in vivo. Notably, AmNA-ASO was efficiently delivered into the mouse brain by intracerebroventricular injection without the aid of additional chemicals. Furthermore, administration of AmNA-ASO ameliorated neurological defects in PD model mice expressing human wild type SNCA. Taken together, these findings suggest that AmNA-ASO is a promising therapeutic strategy for SNCA-associated pathology in PD.

The nucleic acids of ASO can be modified to acquire high nuclease resistance and efficient binding affinities toward complementary strands 23,24 . We have shown that ASOs with amido-bridged nucleic acid (AmNA), an analog of locked nucleic acid (LNA) with modification of the amide bond bridged between the 2′ and 4′ carbons of the ribose, show higher knockdown efficiency and safety compared to natural ASO and LNA [25][26][27] (Supplemental Fig. 1a).
In the present study, our designed AmNA-ASO significantly reduced human SNCA (hSNCA) mRNA and protein levels in human cultured cells and in mice. After administering AmNA-ASO into the intracerebroventricular space of the mouse brain, AmNA-ASO was widely distributed throughout the brain and efficiently taken up by neuronal and, to a lesser extent, non-neuronal cells without the aid of additional chemicals. Moreover, a single injection of AmNA-ASO ameliorated the defects observed in transgenic mice expressing wild type SNCA. Hence, this work highlights the potential of SNCA-targeted AmNA-ASO therapy for PD.

Results
AmNA-ASO efficiently reduced SNCA mRNA levels in human cultured cells. To determine the potency of AmNA-ASO for reducing the levels of SNCA mRNA, we generated a series of AmNA-ASOs (n = 50) covering 80.7% of the coding sequence of SNCA mRNA. The AmNA-ASOs were designed to be 15-mer chimeric antisense oligonucleotides (gapmer) containing AmNA. Supplemental Fig. 1b illustrates the designed AmNA-ASO, which contains AmNA at each end flanking the central bases of DNA with a gapmer motif of 3AmNA-9DNA-2AmNA-1DNA (3-9-2-1).
We screened synthesized AmNA-ASOs for knockdown efficiency of SNCA mRNA in human embryonic kidney 293 (HEK293) cells that express hSNCA mRNA endogenously. For screening, we transfected AmNA-ASOs into HEK293 cells at a single concentration (50 nM) and quantified the SNCA mRNA level using quantitative polymerase chain reaction (qPCR) 24 hours after transfection. We found that several AmNA-ASOs significantly reduced SNCA mRNA levels. In particular, AmNA-ASO No.19 significantly decreased the SNCA mRNA level to 24.5% of the normal expression level in mock transfected cells (control = 100% ± 8.52%, AmNA-ASO No.19 = 24.5% ± 2.29%, p < 0.01 by Dunnett's test), suggesting that AmNA-ASO No. 19 is highly potent for targeting SNCA mRNA in human cultured cells (Fig. 1a). To determine the most efficient construct of AmNA-ASO No. 19, we generated AmNA-ASOs No. 19 with variable lengths and gapmer motif modifications. As a control, we used phosphate-buffered saline (PBS) and scr-AmNA containing the same base composition as AmNA-ASOs No. 19 but in a scrambled order (Fig. 1b). We then transfected the ASOs into HEK293 cells and measured the level of SNCA mRNA expression by qPCR 24 hours after transfection. We found that all of the tested AmNA-ASOs significantly reduced the SNCA mRNA level. AmNA-ASO No. 19 with the gapmer motif of 3AmNA-9DNA-2AmNA-1DNA (3-9-2-1), the same sequence used for the screening described above, was the most efficient and downregulated the SNCA mRNA level to 19.0%, suggesting that AmNA-ASO No. 19 3-9-2-1 (hereinafter referred to as ASO A19 ) is highly potent for targeting SNCA mRNA in the human cultured cells (Fig. 1c).
AmNA-ASOs were shown to be less toxic to animals than LNA-ASOs 27 . To compare the knockdown efficiencies of AmNA-and LNA-ASOs, we generated LNA-ASO No. 19 3-9-2-1 (hereinafter referred to as ASO L19 ) carrying the same target sequence as ASO A19 . We transfected ASO A19 and ASO L19 into HEK293 cells and measured the levels of SNCA mRNA expression by qPCR 24, 72, and 96 hours after transfection, as described above. We found that ASO A19 reduced the SNCA mRNA levels to 10.3% (24 h) and 15.2% (72 h), whereas ASO L19 reduced the levels to 18.3% (24 h) and 28.1% (72 h), suggesting that ASO modified with AmNA knocked down SNCA mRNA more efficiently than that modified with LNA (p = 0.0076, one-way ANOVA, Tukey post hoc test) (Fig. 2a). To evaluate the off-target effects of ASO A19 , we measured the levels of β-synuclein (SNCB) and γ-synuclein (SNCG), which are members of the synuclein family. We found no significant difference in mRNA levels between control and ASO A19 -treated cells, suggesting no off-target effects of ASO A19 on SNCB and SNCG (Fig. 2b). We also evaluated SNCA protein levels by Western blotting 24, 48, 72, and 96 hours after transfection. ASO A19 reduced the expression levels of SNCA protein to similar levels as ASO L19 at both 72 and 96 hours after transfection (Fig. 2c,d and Supplemental Fig. 2), although protein levels were not yet reduced at 24 and 48 hours (Supplemental Fig. 3). Taken together, these data indicate that ASO A19 can reduce SNCA mRNA and protein levels in human cultured cells and that the potency of ASO A19 is comparable to or more efficient than that of ASO L19 . Aso A19 was efficiently delivered into mouse brains, unassisted by transfection reagents. To establish a future ASO treatment for PD, we determined if ASO A19 was effective for targeting SNCA in PD model mice. Intrathecal administration of ASO has recently been established for the treatment of patients affected with SMA 20 . To determine if ASO A19 can be delivered from cerebrospinal fluid (CSF) space to neurons in the central nervous system (CNS), we injected ASO A19 conjugated with fluorescent Alexa 488 into the left lateral ventricle of mice. We extracted the mouse brains 48 hours after injection and examined the localization of ASO. Auto-fluorescence images showed that ASO A19 conjugated with Alexa 488 was widely distributed in the CNS after intracerebroventricular injection (Fig. 3a). Immunofluorescence images showed that ASO A19 was localized in tyrosine hydroxylase (TH) positive neurons in the substantia nigra (arrow in Fig. 3c-c") and neurons surrounded by TH-positive axons in the striatum (arrow in Fig. 3e-e"), suggesting that ASO A19 could be efficiently delivered to TH-and non-TH-positive neurons in vivo (Fig. 3b-e). To further characterize the cell types that take up ASO A19 , we immunostained the mouse brain sections for NeuN and MAP2 (neuronal markers), GFAP (astrocytes marker), O4 (oligodendrocytes marker), and Iba1 (microglial marker). ASO A19 was mainly taken up by neuronal cells (arrow in Fig. 4a,b) and, to some extent, by oligodendrocytes, astrocytes, and microglial cells (arrow in Fig. 4c-e). Furthermore, we evaluated the brain distribution of ASO A19 and found ASO A19 in various brain areas www.nature.com/scientificreports www.nature.com/scientificreports/ including cortex, olfactory bulb, hippocampus, dentate gyrus, striatum, substantia nigra, cerebellum, and brain stem (Fig. 5). These results suggest that ASO injected into the left ventricle is distributed into various cell types and brain areas without the aid of chemical reagents.
Aso A19 efficiently targeted SNCA in transgenic mice expressing hSNCA. To determine if ASO A19 injected into the ventricle could reduce hSNCA expression in the CNS, we tested its effects in the transgenic PD mouse model TH-SNCA-140 m. TH-SNCA-140 m mice carry the hSNCA gene with an Ala53Thr mutation, the expression of which is driven by the TH promoter 28 . We injected 100 μg of control ASO (ASO containing scrambled sequences) and ASO A19 into the left lateral ventricles of two-month-old TH-SNCA-140 m transgenic mice in a single application. Neither injection caused any toxicity-related changes in general health status or behavior. Two weeks after injection, we extracted mRNA from the left cerebral hemisphere and measured hSNCA mRNA levels. Quantification by qPCR showed that ASO A19 significantly decreased hSNCA mRNA levels relative to control ASO (100% (control) vs. 47.2% (ASO A19 ), p < 0.05, t-test, n = 5), which suggests that ASO A19 injected into the CSF can reduce hSNCA mRNA levels in the brain (Fig. 6a). To evaluate the off-target effects of ASO A19 , we measured the levels of endogenous α-synuclein (Snca), β-synuclein (Sncb), and γ-synuclein (Sncg) in mice. We found no significant differences in mRNA levels of these substances between control and ASO A19 -treated mice, suggesting that ASO A19 has no off-target effects (Fig. 6b).
We also evaluated hSNCA protein levels in the right cerebral hemisphere by Western blotting two weeks after injection. ASO A19 significantly reduced the expression levels of SNCA protein (Fig. 6c,d and Supplemental Fig. 4). www.nature.com/scientificreports www.nature.com/scientificreports/ Taken together, these data indicate that ASO A19 can reduce SNCA mRNA and protein levels in the brains of TH-SNCA-140 m transgenic mice.

AmNA-ASO ameliorated defects in motor behavior observed in hSNCA transgenic mice.
To further confirm the efficacy of ASO A19 , we tested it in another transgenic mouse line Thy1-SNCA, which carries human wild type SNCA driven by the murine thymus cell antigen 1 (Thy1) promoter. High levels of hSNCA expression are present in the neocortex, hippocampus, olfactory bulb, thalamus, colliculi, substantia nigra, and brainstem of Thy1-SNCA mice 29 . Moreover, Thy1-SNCA mice have a key feature of PD: high levels of SNCA phosphorylated at serine 129, which is shown to be the predominant form of SNCA found in Lewy bodies, in the substantia nigra, striatum, cortex, frontal cortex, and hippocampus 30 .
In this mouse model, we quantified expression levels of hSNCA protein extracted from left cerebral hemisphere by enzyme-linked immunosorbent assay (ELISA), which is quantitatively sensitive to detect changes in the level of highly expressed hSNCA protein by the Thy1 promoter. We found that hSNCA protein appeared to partition primarily into the Triton X-100 soluble fraction, in contrast to the 10-fold smaller fraction of hSNCA protein in the insoluble fraction. ELISA showed that the levels of SNCA protein in ASO A19 -treated mice were www.nature.com/scientificreports www.nature.com/scientificreports/ significantly decreased in both the soluble and insoluble fractions (soluble hSNCA protein in ASO-injected mice/control = 87.3% ± 2.9%, p = 0.006, t-test and insoluble hSNCA protein in ASO-injected mice/control = 81.5% ± 3.6%, p = 0.0073, t-test), indicating that ASO A19 efficiently reduced hSNCA protein levels in the brains of Thy-1 SNCA mice (Supplemental Fig. 5). Histological evaluation of brain sections from wild type, PBS-, and ASO A19 -injected Thy-1 SNCA mice four weeks after treatment with an antibody specific to hSNCA further confirmed SNCA protein knockdown in ASO A19 -injected mice of the 3 groups (Supplemental Fig. 6). In addition, to assess the effect of SNCA knockdown on the formation of inclusions, we immunostained the brain sections for phosphorylated SNCA, which is frequently used as an indicator for SNCA inclusions. Partial decrease of phosphorylated SNCA density was seen in the cortex, dentate gyrus, and olfactory bulb of ASO A19 -injected Thy-1 SNCA mice (Fig. 7).
Thy-1 SNCA mice also show significant impairments in motor performance at the age of two months, which can be reversed by the dopamine precursor L-dopa, similar to PARK4 and other PD patients [29][30][31] . To determine if ASO A19 could ameliorate behavioral defects in Thy1-SNCA mice, we examined motor behavior in control and ASO A19 -injected mice using the wire suspension test every seven days after injection (See Material and Method). We found that mice injected with ASO A19 showed better performance than control Thy1-SNCA mice. The mean test time for ASO A19 -injected mice was 67.1 s on day 27 after injection, whereas the mean time was 48.2 s for control mice (Fig. 8a). In addition, mice injected with ASO A19 tended to show improvement in the beam walk test, which was performed 28 days after injection using two beams with different widths. We measured the frequency of slips from the beam and normalized this value to the walking speed along the beams. ASO A19 -injected mice showed tendency of reduced frequency of slips per speed compared to controls (ASO A19 vs. control: 168.7 vs 378.8 per m/s (beam 1) and 248.8 vs. 438.1 per m/s (beam 2), Fig. 8b). Lastly, we examined the effects of ASO A19 on the pasta gnawing test, which can sensitively evaluate eating behavior that requires fine coordination of limb and oromotor functions [32][33][34] . Notably, ASO A19 -injected mice showed significantly better performances than control mice. On day 20 after injection, the mean number of bites per episode was 3.94 for control mice and 6.44 for ASO A19 -injected mice (p = 0.0051, t-test). On day 27 after injection, the mean number of bites per episode was 3.92 for control mice and 6.8 for ASO A19 -injected mice (p = 0.0021, t-test) (Fig. 8c). Taken together, these data indicate that ASO A19 can improve behavioral defects observed in Thy1-SNCA mice accompanied by downregulation of SNCA. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In the present study, we generated ASO A19 capable of reducing the level of hSNCA mRNA in HEK293 cells and found that ASO A19 is superior to ASO L19 for decreasing hSNCA mRNA. Further, we confirmed that ASO A19 was widely distributed in the CNS following a single intracerebroventricular administration. This study is the first to report that intracerebroventricular administration of ASO in the absence of any carrier or conjugation can knockdown the level of hSNCA and improve motor dysfunction in transgenic animal PD models.
SNCA protein is one of the most promising therapeutic targets for the treatment of PD 35 . We showed that ASO A19 can target wild type and mutant hSNCA, indicating its applicability for both familial and sporadic PD. Moreover, reducing the level of SNCA expression is likely to protect against neuronal toxicities. We previously reported that reduction of SNCA expression enabled the survival of more TH-positive neurons in 1-methyl-4 -phenyl-1,2,3,6-tetrahydropyridine model rats 36 . SNCA knockdown by shRNA attenuates the progression of motor deficits in rotenone-exposed rats 37 , suggesting that reduction of SNCA expression might also be beneficial for PD. Benskey et al. reported that excessive reduction of SNCA within nigrostriatal neurons induced rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) and resulted in the death of affected neurons 38 . However, several studies have shown that normalization of overexpressed SNCA decreased neurodegeneration of dopaminergic neurons and improved motor function in animal models 37,39 . These data imply that adequate downregulation of SNCA can prevent neurodegeneration with little disadvantages.
To improve the efficacy of antisense oligonucleotides for in vivo therapeutic application, we developed a less toxic ASO containing AmNA 25,26 . We then applied AmNA-ASO targeting Y-box binding protein-1 effectively as an antiangiogenic cancer therapy 27 . To increase ASO stability in vivo and reduce the number of treatments, we applied the technique of AmNA modification in ASO targeting hSNCA. Importantly, AmNA modification enhanced the potency of ASO for targeting SNCA (Fig. 2a-d). Consistently, single intracerebroventricular administration of ASO A19 was effective for targeting SNCA in the mouse CNS even two weeks after injection (Fig. 6c,d), supporting the stability of intracerebroventricularly administered ASO A19 in patients with α-synucleinopathies. www.nature.com/scientificreports www.nature.com/scientificreports/ In an alternative approach to targeting SNCA protein, antibodies against the C-terminus of SNCA protein were shown to reduce accumulation of C-terminal truncated SNCA protein, prevent TH loss in the striatum, and improve motor deficits in SNCA transgenic mouse models 40 . A phase I study of administration of this anti-SNCA antibody, PRX002, was performed and showed that serum SNCA protein could be modulated without any Figure 5. Widespread distribution of ASO A19 throughout the mouse brain. Immunostaining images of brain sections collected from Alexa 488 conjugated-ASO A19 -administered wild type mice. Widespread distribution of ASO A19 in different brain areas, including the cortex, olfactory bulb, hippocampus, dentate gyrus, striatum, substantia nigra, cerebellum, and brain stem, was seen. Merged images showing AmNA-Alexa488 in green and DAPI-labeled cell nuclei in blue. Scale bar: 20 μm.
www.nature.com/scientificreports www.nature.com/scientificreports/ serious adverse events 41 . However, this anti-SNCA immunotherapy works primarily by recognizing and clearing extracellular SNCA protein to block cell-to-cell propagation of SNCA in the brain and is less effective at targeting intraneuronal SNCA. ASO A19 administration could be a complementary approach used with anti-SNCA immunotherapy.
Although we found no off-target or toxic effects of ASO A19 (Fig. 6b) (Supplemental Fig. 7), the safety of ASO A19 injection must be confirmed for future clinical applications. Studies using SNCA knock-out mice showed that SNCA might be required for synaptic function 42,43 and maintenance of subependymal neural stem cells 44 . It is important to note that antisense therapy does not cause total loss of SNCA expression (Fig. 6a). Hence, a modest reduction of SNCA expression is not likely to cause serious defects, even if SNCA is required for some biological functions in the human brain.
In conclusion, we demonstrated that intracerebroventricular administration of AmNA-ASO can reduce SNCA mRNA and the corresponding protein levels, resulting in improvement of some motor dysfunctions observed in PD model mice. Remarkably, intracerebroventricular administration of nusinersen, an ASO for treating SMA, was recently approved [19][20][21][22] . Our findings highlight the potential of AmNA-ASO as a novel therapy for PD and other synucleinopathies. www.nature.com/scientificreports www.nature.com/scientificreports/

Material and Methods
Oligonucleotides. The ASOs used in this study, which were synthesized by GeneDesign, are gapmer-type 13-15-mer phosphorothioate oligonucleotides containing five AmNA modifications (Supplemental Fig. 1b).
AmNA-ASOs containing specific DNA sequences (TCC and TGC), which have been shown to bind to hepatocellular proteins and cause hepatotoxicity 45 , induce cellular responses mediated by Toll-like receptor 9, and cause strong inflammatory responses (CpG), were excluded 46 . The loop structure of SNCA mRNA, where ASOs easily bind target RNA, was predicted using the mFold web server 47 and the 50 most effective sequences targeting hSNCA mRNA were selected.

Cell culture and transfection. HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco's Modified
Eagle Medium (Sigma) supplemented with 10% fetal bovine serum at 37 °C in a humidified chamber of 95% O 2 and 5% CO 2 . Cells were plated into 12-well plates 24 hours before transfection. AmNA-ASOs were transfected into HEK293 cells with Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocols. The final concentration of AmNA-ASOs was adjusted to 50 nM. All ASOs were tested in triplicate.
Quantitative RT-PCR of total RNA extracted from HEK293 cells and mouse brains. Total RNA was extracted from HEK293 cells using a RNeasy Mini kit (QIAGEN). RNA was reverse transcribed to complementary DNA (cDNA) using the Superscript III First Strand cDNA system (Life Technologies) according to the manufacturer's protocol and cDNA was used as the template for qPCR. qPCR was performed using TaqMan Gene Expression assays on an ABI PRISM 7900HT Sequence Detection System (Life Technologies). Relative mRNA expression was normalized by the 18 s ribosomal RNA level in each sample and calculated using the delta-delta Ct method. Taqman primer/probe sets specific for hSNCA, hSNCB, hSNCG, mouse Snca, mouse Sncb, mouse Sncg, and 18 s ribosomal RNA were purchased from Thermo Fisher Scientific.
To quantify hSNCA mRNA levels in mice, brain samples were collected and separated into left and right cerebral hemispheres. Left hemispheres were used for qPCR and right hemispheres for Western blot analysis. Left Immunohistological images of various brain areas in wild type, control, and ASO A19 -treated Thy-1 SNCA mice using a monoclonal antibody that recognizes phosphorylated SNCA. ASO A19 -treated Thy1-SNCA mice showed partial reduction of phosphorylated SNCA in the cortex, olfactory bulb, and dentate gyrus relative to control mice (arrow). Scale bars: 100 μm. (2019) 9:7567 | https://doi.org/10.1038/s41598-019-43772-9 www.nature.com/scientificreports www.nature.com/scientificreports/ hemispheres were immediately snap-frozen in liquid nitrogen and stored at −80 °C. For analysis, frozen left brain hemispheres were ground using a prechilled mortar and pestle with liquid nitrogen occasionally added to prevent thawing. After the tissue was finely ground, ISOGEN (Nippon gene) was added to isolate total RNA, according to the manufacturer's instructions. Reverse transcription and qPCR were performed as described above.
Western blot analysis of SNCA protein from HEK293 cells and mouse brains. Proteins were extracted from HEK293 cells using the CelLytic M Cell Lysis Reagent (Sigma-Aldrich) with protease and phosphatase inhibitor cocktail. Protein concentration was measured using a Pierce ™ BCA Protein Assay Kit (Thermo Fisher Scientific). Cell lysates were mixed with Laemlli sample buffer, boiled, and separated on polyacrylamide gel. After protein transfer to polyvinylidene fluoride membranes, the blots were probed with the primary antibodies, α-synuclein (BD Transduction Laboratories, cat #610787, 1:10000 dilution) and actin (Millipore, cat #MAB1501, 1:1000 dilution); visualized with secondary antibodies, HRP-conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare); and developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare). The signals of immunoblots were acquired with a Chemidoc Touch Imaging System (Bio-Rad).
To quantify hSNCA protein in mouse brains, proteins were extracted from the right cerebral hemispheres using the CelLytic ™ MT Cell Lysis Reagent for mammalian tissues (Sigma-Aldrich) with protease and phosphatase inhibitor cocktail. The BCA assay and Western blotting were performed as described above. The primary antibody used for hSNCA detection was α-synuclein (211) (Santa Cruz, cat #sc-12767, 1:1000 dilution).
Mouse strains. TH-SNCA-140 m transgenic mice with a C57BL/6J background carrying the hSNCA gene with a Ala53Thr mutation, for which expression is driven by the TH promoter 28 , and Thy1-SNCA transgenic mice (Line 61) with a C57BL/6 × DBA/2F1 background carrying human wild type SNCA driven by the murine thymus cell antigen 1 promoter 29-31 were used. www.nature.com/scientificreports www.nature.com/scientificreports/ Animal housing. Mice were housed in individual ventilated cages. Each cage contained a maximum of four mice of the same sex. Mice were housed under a 12 hours light/dark cycle and standard housing conditions with ad libitum access to food and water for one week before the experiment. All animal experiments were conducted according to ARRIVE guidelines.

Intracerebroventricular injection of AmNA-ASOs into mice.
Mice were anesthetized with an intraperitoneal injection of cocktails comprising medetomidine (2 mg/kg), midazolam (10 mg/kg), butorphanol (1 mg/kg) or ketamine (50 mg/kg), medetomidine (0.3 mg/kg), and butorphanol (5 mg/kg). Anesthetized mice were placed into a stereotaxic frame and a single burr hole was drilled to allow placement of the stereotactic needle (Hamilton ® ). The coordinates for injection were 0.3 mm anterior and 1.0 mm lateral to Bregma and 3 mm below the dura. AmNA-ASOs dissolved in 10 μl phosphate-buffered saline (PBS) were injected into the left lateral ventricle at a rate of 2 μl/min 48 .
Behavioral testing. For mice administered with AmNA-ASOs, body weight was measured and pasta gnawing test 32 and wire suspension test 49 were performed four times per week. The beam walk test 50,51 was performed once after the final pasta gnawing and wire suspension tests. For the pasta gnawing test, mice were brought into the experimental room at least two hours before testing. The cage top, water bottle, and food pellets were removed (only the cage lid remained) and a small piece of dry spaghetti (approximately 5 mm) was placed in the cage. After habituation, the home cage was placed on the testing table. Spaghetti noodles were broken into several pieces approximately 1 cm long and placed in the center of the cage. A microphone was placed above the noodle pieces and recording was initiated as soon as the animal started to eat. The number of bites per gnawing episode and biting frequency were evaluated. The acquired gnawing pattern data were analyzed using Avisoft SASLab Pro.
For the wire suspension test, a wire cage lid was used with duct tape placed around the perimeter to prevent the mouse from walking off the edge. The animal was placed on top of the cage lid and the lid was lightly shaken three times to force the mouse to grip the wires. The lid was then turned upside down and held approximately 50-60 cm above a soft underlay, high enough to prevent the mouse from jumping down but not high enough to cause harm in the event of a fall. The latency to fall was quantified.
For the beam walk test, we used long strips of wood (beams) with a 1000 × 13 or 10 mm cross-section. The beams were placed horizontally 50 cm above the surface, with one end mounted on a narrow support and the other end attached to the home cage into which the mouse could escape. A desk lamp was positioned above the start of the beam. Three training trials were performed prior to testing using three different starting points on the 20 mm square beam: close proximity to the home cage (trial 1), the center of the beam (trial 2), and at the brightly illuminated end of the beam (trial 3). After the mice were trained, they underwent test trials on each of the square beams. The test trials were videotaped and then evaluated with Observer XT 10.5 (Noldus). The latency to traverse each beam and the number of times the hind feet slipped off each beam were recorded for each trial.

Histological analysis of SNCA protein in mouse brains. Brain samples were taken from PBS-and
AmNa-ASO-treated Line 61 mice and wild type littermates. The hemispheres were divided at the midline. One hemisphere was fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 2 hours at RT, cryoprotected in 15% sucrose in PBS overnight, embedded in OCT medium in cryomolds, and snap-frozen in dry ice-cooled liquid isopentane. Frozen samples were stored at −80 °C until sectioning. Frozen brain hemispheres were cryosectioned sagittally at 30 µm thickness on a Leica CM1850. Sections were transferred to 30% sucrose solution in PBS (Thermo Scientific) and stored at 4 °C. For immunostaining, sections were washed and incubated with primary and secondary antibodies as previously described in Furuya et al. 52 . In brief, the following primary antibodies were used: phosphorylated α-synuclein antibody, a mouse monoclonal antibody against α-synuclein phosphorylated at Ser129 (Wako, cat # 015-25191, 1:1000 dilution) and Syn211 (Invitrogen, AHB0261, 1:1000 dilution), and a mouse monoclonal antibody that specifically recognizes human α-synuclein. After immunostaining, mounted sections were observed and imaged under a fluorescence microscope (BZ-X700, Keyence). ELISA for SNCA protein expression. Mouse brains were collected 30 days after injection. Mashed left brain hemispheres were homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1% Triton X-100, 0.2 mM Sodium-orthovanadate, protease inhibitor cocktail (Calbiochem), and phosphatase inhibitor cocktail (Sigma)). Homogenates were incubated for 30 min on ice, followed by centrifugation at 15,000 g for 60 min at 4 °C. Supernatant was collected as the Triton X-100 soluble fraction. The Triton X-100-insoluble pellet was washed once in lysis buffer and then dissolved in lysis buffer containing 2% SDS. The resulting homogenate in 2% (2019) 9:7567 | https://doi.org/10.1038/s41598-019-43772-9 www.nature.com/scientificreports www.nature.com/scientificreports/ SDS was collected as the Triton X-100 insoluble fraction. hSNCA protein levels in the Triton X-100 soluble and insoluble fractions of all mice were determined using a commercially-available immunosorbent assay (Meso Scale Discovery, Cat#. K151TGD-4; Lot# K00E0147) according to the manufacturer's protocol. Plates were read on the Sector Imager 2400. For the assay, Triton X-100 soluble fractions were diluted 1:20000 and insoluble fractions 1:2000 in assay buffer. SNCA protein levels were evaluated in comparison to an adequate peptide standard as μg hSNCA and normalized to tissue weight (wet weight).

Statistical analysis.
Dunnett's test was used to compare several treatments with a single control (e.g. Fig. 1a). One-way ANOVA, Tukey post hoc test was performed to compare the efficacy of control, ASO A19 , and ASO L19 in HEK293 cells. Two-tailed unpaired t-tests were performed to compare the efficacy of control vs ASO A19 in mice. For all figures, the error bars represent the standard error of the mean (SEM). P-values below 0.05 were considered statistically significant. Study approval. This research was approved by the institutional ethics committee. All of the procedures for animal experiments were approved by the institutional animal care and use committee of Osaka University.