Artigo Acesso aberto Revisado por pares

The AKT modulator A-443654 reduces α-synuclein expression and normalizes ER stress and autophagy

2021; Elsevier BV; Volume: 297; Issue: 4 Linguagem: Inglês

10.1016/j.jbc.2021.101191

ISSN

1083-351X

Autores

Mandi Gandelman, Warunee Dansithong, Stephen C. Kales, Sharan Paul, Gentrie Maag, Erika Aoyama, Alexey Zakharov, Ganesha Rai, Thomas S. Dexheimer, Brooke M. Whitehill, Hongmao Sun, Ajit Jadhav, Anton Simeonov, Mark J. Henderson, Duong P. Huynh, Stefan M. Pulst, Daniel R. Scoles,

Tópico(s)

Autophagy in Disease and Therapy

Resumo

Accumulation of α-synuclein is a main underlying pathological feature of Parkinson's disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson's disease pathology. Accumulation of α-synuclein is a main underlying pathological feature of Parkinson's disease and α-synucleinopathies, for which lowering expression of the α-synuclein gene (SNCA) is a potential therapeutic avenue. Using a cell-based luciferase reporter of SNCA expression we performed a quantitative high-throughput screen of 155,885 compounds and identified A-443654, an inhibitor of the multiple functional kinase AKT, as a potent inhibitor of SNCA. HEK-293 cells with CAG repeat expanded ATXN2 (ATXN2-Q58 cells) have increased levels of α-synuclein. We found that A-443654 normalized levels of both SNCA mRNA and α-synuclein monomers and oligomers in ATXN2-Q58 cells. A-443654 also normalized levels of α-synuclein in fibroblasts and iPSC-derived dopaminergic neurons from a patient carrying a triplication of the SNCA gene. Analysis of autophagy and endoplasmic reticulum stress markers showed that A-443654 successfully prevented α-synuclein toxicity and restored cell function in ATXN2-Q58 cells, normalizing the levels of mTOR, LC3-II, p62, STAU1, BiP, and CHOP. A-443654 also decreased the expression of DCLK1, an inhibitor of α-synuclein lysosomal degradation. Our study identifies A-443654 and AKT inhibition as a potential strategy for reducing SNCA expression and treating Parkinson's disease pathology. Parkinson's disease (PD) is the second most common neurological disorder, and currently there are no treatments that modify disease progression. Mutations in the SNCA gene can cause autosomal dominant Parkinson's disease 1 (PARK1), and duplications or triplications of wildtype SNCA are also associated with sporadic PD cases (1Chartier-Harlin M.C. Kachergus J. Roumier C. Mouroux V. Douay X. Lincoln S. Levecque C. Larvor L. Andrieux J. Hulihan M. Waucquier N. Defebvre L. Amouyel P. Farrer M. Destee A. Alpha-synuclein locus duplication as a cause of familial Parkinson's disease.Lancet. 2004; 364: 1167-1169Abstract Full Text Full Text PDF PubMed Scopus (1475) Google Scholar, 2Ibanez P. Bonnet A.M. Debarges B. Lohmann E. Tison F. Pollak P. Agid Y. Durr A. Brice A. Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease.Lancet. 2004; 364: 1169-1171Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 3Singleton A.B. Farrer M. Johnson J. 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Aggregated and misfolded α-synuclein is the main component of Lewy bodies occurring in the substantia nigra pars compacta, which are a hallmark feature of PD, and is associated with dementia with Lewy bodies, diffuse Lewy body disease, and glial cytoplasmic inclusions in multiple system atrophy collectively known as the synucleinopathies (6Goedert M. Jakes R. Spillantini M.G. The synucleinopathies: Twenty years on.J. Parkinsons Dis. 2017; 7: S51-S69Crossref PubMed Scopus (201) Google Scholar). Evidence of SNCA multiplications and constitutive phosphorylation in synucleinopathies suggests overexpression and toxic gain of function as a causative factor of disease, supported by increased neuronal cell death being associated with altered SNCA expression in patients with PD and in model systems (7Chiba-Falek O. Lopez G.J. Nussbaum R.L. Levels of alpha-synuclein mRNA in sporadic Parkinson disease patients.Mov. 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Seeking a mechanism for the toxicity of oligomeric alpha-synuclein.Biomolecules. 2015; 5: 282-305Crossref PubMed Scopus (135) Google Scholar). Moreover, dissociation of fibrillar α-synuclein may lead to elevation of toxic α-synuclein oligomers, leading to uncertainty about which α-synuclein species is best to target to reduce toxicity (15Bengoa-Vergniory N. Roberts R.F. Wade-Martins R. Alegre-Abarrategui J. alpha-Synuclein oligomers: A new hope.Acta Neuropathol. 2017; 134: 819-838Crossref PubMed Scopus (150) Google Scholar, 16Zhang G. Xia Y. Wan F. Ma K. Guo X. Kou L. Yin S. Han C. Liu L. Huang J. Xiong N. Wang T. New perspectives on roles of alpha-synuclein in Parkinson's disease.Front. Aging Neurosci. 2018; 10: 370Crossref PubMed Scopus (48) Google Scholar). Ultimately, evidence indicates that an approach for lowering the overall expression of α-synuclein would be promising as a therapeutic for PD and other α-synucleinopathies. Studies using animal models support that therapeutics lowering overall SNCA expression may improve PD phenotypes, even when treatment begins after disease onset: In a mouse model of Lewy body disease, with inducible A53T-SNCA expression, transgene suppression improved learning and memory phenotypes and reduced Lewy body-like aggregates in cortex and hippocampus (17Lim Y. Kehm V.M. Lee E.B. Soper J.H. Li C. Trojanowski J.Q. Lee V.M. alpha-Syn suppression reverses synaptic and memory defects in a mouse model of dementia with Lewy bodies.J. Neurosci. 2011; 31: 10076-10087Crossref PubMed Scopus (83) Google Scholar). Similarly, in A30P-SNCA inducible mice, abnormal olfactory bulb dopamine levels and behavioral deficits were restored by transgene suppression (18Nuber S. Petrasch-Parwez E. Arias-Carrion O. Koch L. Kohl Z. Schneider J. Calaminus C. Dermietzel R. Samarina A. Boy J. Nguyen H.P. Teismann P. Velavan T.P. Kahle P.J. von Horsten S. et al.Olfactory neuron-specific expression of A30P alpha-synuclein exacerbates dopamine deficiency and hyperactivity in a novel conditional model of early Parkinson's disease stages.Neurobiol. Dis. 2011; 44: 192-204Crossref PubMed Scopus (21) Google Scholar). In order to identify small molecule SNCA inhibitors we performed a quantitative high-throughput screen to identify compounds lowering SNCA expression. The screening assay was generated using genome editing to insert the luciferase gene downstream and in frame of the SNCA gene in HEK-293 cells (19Dansithong W. Paul S. Scoles D.R. Pulst S.M. Huynh D.P. Generation of SNCA cell models using zinc finger nuclease (ZFN) technology for efficient high-throughput drug screening.PLoS One. 2015; 10e0136930Crossref PubMed Scopus (13) Google Scholar). We screened 155,885 compounds and identified multiple hits, including a known AKT inhibitor A-443654. A-443654 lowered SNCA expression in PD patient–derived fibroblasts and in dopaminergic neurons differentiated from PD patient–derived induced pluripotent stem cells (iPSCs). In addition, A-443654 normalized α-synuclein monomers and oligomers, SCNA expression, and autophagy and ER stress markers in HEK-293 cells. Our study identified A-443654 as a small molecule regulator of α-synuclein levels and toxicity in multiple cellular models of PD. Previously we engineered a cell line that expresses luciferase from the SNCA locus, HEK-293-SNCA-luc, which demonstrated robust response to SNCA siRNA or a small molecule luciferase inhibitor (19Dansithong W. Paul S. Scoles D.R. Pulst S.M. Huynh D.P. Generation of SNCA cell models using zinc finger nuclease (ZFN) technology for efficient high-throughput drug screening.PLoS One. 2015; 10e0136930Crossref PubMed Scopus (13) Google Scholar). To further validate that luciferase activity specifically reflects transcriptional activity from the SNCA locus, we performed a clustered regularly interspaced short palindromic repeat (CRISPR) interference (CRISPRi) assay. A single guide RNA was used to direct a nuclease deactivated Cas9 (dCas9) tethered to the KRAB transcriptional repressor to just downstream of the SNCA transcriptional start site (TSS) (20Gilbert L.A. Larson M.H. Morsut L. Liu Z. Brar G.A. Torres S.E. Stern-Ginossar N. Brandman O. Whitehead E.H. Doudna J.A. Lim W.A. Weissman J.S. Qi L.S. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.Cell. 2013; 154: 442-451Abstract Full Text Full Text PDF PubMed Scopus (1954) Google Scholar). Cotransfection of CRISPRi plasmids in HEK-293-SNCA-luc cells resulted in 45% reduction of luciferase expression from the SNCA locus compared with untransfected cells, whereas transfection of CRISPRi plasmids in HEK-293-CMV-luc control cells did not inhibit luciferase expression (Fig. S1). We screened a total of 155,885 compounds, with 3 to 11 doses per compound depending on how the libraries were originally formatted, and measured the effects on SNCA-luc expression (21Inglese J. Auld D.S. Jadhav A. Johnson R.L. Simeonov A. Yasgar A. Zheng W. Austin C.P. Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11473-11478Crossref PubMed Scopus (586) Google Scholar). The primary screen also included a viability measurement, using Gly-Phe-7-amino-4-trifluoromethylcoumarin (GF-AFC), and compounds were eliminated from further consideration if they were cytotoxic. The total number of active compounds passing cytotoxicity (cytotox) triage was 1996 (1.3%). The number of compounds screened, doses, and Z′ factors are provided in Table 1. After in silico analysis (clustering, removal of frequent hitter nuisance chemotypes), 842 compounds were selected for follow-up and retested at 11 doses in the SNCA-luc cells (luciferase and GF-AFC confirmation) and a CMV-luc expressing cell line to flag general transcriptional repressors. Following this triage, 33 compounds were tested by enzyme-linked immunosorbent assay (ELISA) to measure endogenous α-synuclein protein in 3XSNCA PD patient fibroblasts. Triaging of compounds through validation counter-screens and orthogonal assays is shown in Table 2. Five compounds were confirmed to reduce α-synuclein levels in secondary assays and by ELISA using patient cells, including NCGC00470280, NCGC00419055, NCGC00448911, NCGC00345809, and NCGC00347278 (A-443654) (Table 3). We eliminated NCGC00345809 from further consideration for having the least favorable cytotox and ELISA parameters (Table 3). Among the remaining compounds, A-443654 displayed the most promising profile when primary assays, ELISA, and Western blot results were considered collectively and was selected for further study. The structure of A-443654 is shown in Figure 1A. General transcriptional effects were not observed in the CMV-luc counter-screen (IC50 8.91 μM) (Fig. 1B).Table 1Libraries, compound numbers, doses, Z′ factors, activitiesLibraryCompoundsDosesZ' (SNCA-luc)Active (SNCA-luc)Passing cytotox triageNPCaThe NPC library included only three doses, so curve classes −1.1, −1.2, −2.1, −2.2 could not be computed. There were 12 compounds that showed inhibition (all negative curve classes).281630.421210Epigenetics Focused Library33940.5164NPACT515770.59299Genesis96,26040.619371562PTL300040.621411MIPE 4.01978110.6515547LOPAC128040.601815Sytravon45,05540.632719333Sum for all libraries155,885----48901996The total number of compounds was 155,885.a The NPC library included only three doses, so curve classes −1.1, −1.2, −2.1, −2.2 could not be computed. There were 12 compounds that showed inhibition (all negative curve classes). Open table in a new tab Table 2Triage of 842 compounds through validation counter-screens (SNCA-luc, CMV-luc, Cytotox) and orthogonal assays (ELISA)Triage stepCriterionaCriteria for advancing compounds: (1) Negative curve class in primary screen. (2) Confirmed negative curve class, >30% inhibition for SNCA-luc assay. (3) ΔAUC (single metric of potency and efficacy) of >40 comparing SNCA-luc versus both CMV-luc and cytotox assays. (4) >50% reduction for SNCA at 1 μM.No. of CompoundsTotal evaluated in follow-ups1842Confirmed in the SNCA-luc assay (of 842)2446Passing CMV-luc and Cytotox counter-screens (of 446)333Lowering SNCA in 3XSNCA fibroblasts by ELISA (of 33)45a Criteria for advancing compounds: (1) Negative curve class in primary screen. (2) Confirmed negative curve class, >30% inhibition for SNCA-luc assay. (3) ΔAUC (single metric of potency and efficacy) of >40 comparing SNCA-luc versus both CMV-luc and cytotox assays. (4) >50% reduction for SNCA at 1 μM. Open table in a new tab Table 3Lead compoundsCompoundMWFunctionPrimary assay IC50 (μM)SNCA efficacy (%)Curve classCytotox IC50 (μM)aDesignated n/a for inactive cytotox curve class of 4.Cytotox efficacy (%)aDesignated n/a for inactive cytotox curve class of 4.Cytotox curve classELISA IC50bSNCA triplicated human fibroblast cell line ND27760.α-Synuclein reduction at 0.5 μM (WB) (%)cHEK-293 cells.NCGC00470280400.6Unknown15.8−60−2.4n/an/a430 nM82NCGC00419055276.3Unknown15.8−85−1.2n/an/a4366 nM29NCGC00345809473.3Cdk/Crk Inhibitor0.11−74−1.20.09−42−1.2411 nMn.d.dValue not determined.NCGC00448911421.4Unknown15.8−66−1.2n/an/a499 nM67NCGC00347278 (A-443654)397.5AKT inhibitor0.25−86−1.10.89−53−1.289 nM85a Designated n/a for inactive cytotox curve class of 4.b SNCA triplicated human fibroblast cell line ND27760.c HEK-293 cells.d Value not determined. Open table in a new tab The total number of compounds was 155,885. A-443654 is a well-characterized inhibitor of the serine/threonine kinase Akt. Inhibition of Akt by A-443654 is associated with "paradoxical" Akt phosphorylation at Ser473, while effectively inhibiting its downstream effectors GSK3α/β, molecular target of rapamycin (mTOR), and TSC2 (22Luo Y. Shoemaker A.R. Liu X. Woods K.W. Thomas S.A. de Jong R. Han E.K. Li T. Stoll V.S. Powlas J.A. Oleksijew A. Mitten M.J. Shi Y. Guan R. McGonigal T.P. et al.Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo.Mol. Cancer Ther. 2005; 4: 977-986Crossref PubMed Scopus (204) Google Scholar, 23Han E.K. Leverson J.D. McGonigal T. Shah O.J. Woods K.W. Hunter T. Giranda V.L. Luo Y. Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition.Oncogene. 2007; 26: 5655-5661Crossref PubMed Scopus (129) Google Scholar, 24Chan T.O. Zhang J. Rodeck U. Pascal J.M. Armen R.S. Spring M. Dumitru C.D. Myers V. Li X. Cheung J.Y. Feldman A.M. Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: E1120-E1127Crossref PubMed Scopus (57) Google Scholar). In agreement with this, A-443654 increased AKT Ser473 phosphorylation in a dose-dependent manner in HEK-293 cells, which was associated with reduced total AKT protein abundance after extended exposure to the inhibitor (Fig. 2). Decreased phospho-GSK3α/β was observed in response to A-443654, consistent with inhibition of AKT signaling (Fig. 2). To characterize the effects of A-443654 on α-synuclein expression we used HEK-293 cells (ATXN2-Q22) in which the ATXN2 allele was replaced with a pathogenic variant containing a CAG expansion of 58 repeats (ATXN2-Q58 cells). This cell line represents a model of spinocerebellar ataxia type 2 (25Paul S. Dansithong W. Figueroa K.P. Scoles D.R. Pulst S.M. Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration.Nat. Commun. 2018; 9: 3648Crossref PubMed Scopus (44) Google Scholar) useful for our testing because α-synuclein monomers, dimers, and trimers can readily be detected by Western blot (at estimated 17, 34 and 51 kDa, respectively) and its levels are highly increased. It had previously been established that the antibody we used is competent for detecting high-molecular-weight forms of α-synuclein (26Choi I. Zhang Y. Seegobin S.P. Pruvost M. Wang Q. Purtell K. Zhang B. Yue Z. Microglia clear neuron-released alpha-synuclein via selective autophagy and prevent neurodegeneration.Nat. Commun. 2020; 11: 1386Crossref PubMed Scopus (93) Google Scholar, 27Jiang P. Gan M. Yen S.H. McLean P.J. Dickson D.W. Impaired endo-lysosomal membrane integrity accelerates the seeding progression of alpha-synuclein aggregates.Sci. Rep. 2017; 7: 7690Crossref PubMed Scopus (42) Google Scholar, 28Landeck N. Strathearn K.E. Ysselstein D. Buck K. Dutta S. Banerjee S. Lv Z. Hulleman J.D. Hindupur J. Lin L.K. Padalkar S. Stanciu L.A. Lyubchenko Y.L. Kirik D. Rochet J.C. Two C-terminal sequence variations determine differential neurotoxicity between human and mouse alpha-synuclein.Mol. Neurodegener. 2020; 15: 49Crossref PubMed Scopus (1) Google Scholar, 29Kumar V.B. Hsu F.F. Lakshmi V.M. Gillespie K.N. Burke W.J. Aldehyde adducts inhibit 3,4-dihydroxyphenylacetaldehyde-induced alpha-synuclein aggregation and toxicity: Implication for Parkinson neuroprotective therapy.Eur. J. Pharmacol. 2019; 845: 65-73Crossref PubMed Scopus (14) Google Scholar). To assure this, we knocked down SNCA expression by RNAi and observed no monomeric α-synuclein and the greatest reduction of dimeric and trimeric forms at the longest treatment time (Fig. S2). We observed elevated levels of α-synuclein protein monomers and trimers (2.5 ± 0.4-fold increase in monomers and 1.7 ± 0.3-fold increase in trimers) in ATXN2-Q58 cells compared with the isogenic parent cell line with a normal ATXN2 (Fig. 3A). In addition, ATXN2-Q58 cells exhibited transcriptional upregulation of SNCA mRNA (Fig. 3B). We did not quantify α-synuclein dimers because they were of low abundance and exhibited high interexperiment variability. Treatment of ATXN2-Q58 cells with A-443654 for 48 h completely restored the levels of trimer and monomer α-synuclein proteins (Fig. 3A and C) and significantly reduced SNCA mRNA levels (Fig. 3D). We next studied skin fibroblasts from a patient with PD with a triplication of the SNCA gene (3XSNCA fibroblasts). Compared with skin fibroblasts from a healthy individual (normal) or patients with parkinsonism, Gaucher's disease, or PD linked to mutations in the GBA or LRKK2 gene, the 3XSNCA fibroblasts displayed between 1.7- and 2.1-fold increase in α-synuclein abundance (Fig. 4A). 3XSNCA fibroblasts treated with A-443654 for 48 h showed greater reduction of α-synuclein abundance at higher doses, as determined by ELISA (Fig. 4B), without cytotoxic effects (Fig. 4C). Western blotting analysis of 3XSNCA fibroblasts showed no detectable monomeric or dimeric forms of α-synuclein; however, α-synuclein trimers were elevated 3-fold and A-443654 successfully lowered them to levels comparable with normal fibroblasts (Fig. 4D). SNCA mRNA was highly increased in 3XSNCA fibroblasts (20-fold compared with normal control), and 1 μM A-443654 significantly reduced SNCA mRNA abundance by 38% (Fig. 4E). We next examined the activity of A-443654 in dopaminergic neurons, the cell type most impacted in PD. iPSCs with a triplication of the SNCA gene were differentiated into dopaminergic neurons, which were positive for tyrosine hydroxylase and had neurite outgrowths characteristic of neurons (Fig. S3). The PD 3XSNCA neurons had a >5-fold increase in α-synuclein monomer expression compared with normal neurons, and 0.1 μM A-443654 treatment reduced the abundance of monomers by >3-fold after a 48-h treatment (Fig. 5). α-Synuclein dimers and trimers were not detected in these cells (Fig. 5). Accumulation of α-synuclein leads to cellular stress and subsequent activation of signaling that can either promote survival or activate cell death pathways in the presence of overwhelming damage. We have previously described that ATXN2-Q58 cells display significant increases in unfolded protein response (UPR) mediators and autophagic markers, as well as the RNA-binding protein STAU1, which can enhance the detrimental effects of ATXN2 expansions (30Gandelman M. Dansithong W. Figueroa K.P. Paul S. Scoles D.R. Pulst S.M. Staufen 1 amplifies proapoptotic activation of the unfolded protein response.Cell Death Differ. 2020; 27: 2942-2951Crossref PubMed Scopus (12) Google Scholar). Upon treatment with A-443654, decreases in α-synuclein levels were associated with normalized STAU1 abundance (Fig. 6A). In addition, autophagy mediators mTOR, p62, and LC3-II and UPR factors BiP and CHOP returned to baseline levels, indicating A-443654 efficiently restored protein homeostasis (Fig. 6A). The expression of ATXN2 was unchanged, indicating that the effects of A-443654 are independent of ATXN2 (Fig. 6A). Previous studies revealed that knockdown of doublecortin like kinase 1 (DCLK1) reduced α-synuclein level and toxicity (31Vazquez-Velez G.E. Gonzales K.A. Revelli J.P. Adamski C.J. Alavi Naini F. Bajic A. Craigen E. Richman R. Heman-Ackah S.M. Wood M.J.A. Rousseaux M.W.C. Zoghbi H.Y. Doublecortin-like kinase 1 regulates alpha-synuclein levels and toxicity.J. Neurosci. 2020; 40: 459-477Crossref PubMed Scopus (7) Google Scholar). Therefore, we investigated whether A-443654 could modulate DCLK1 in our ATXN2-Q58 model. We found that A-443654 dose dependently decreased the levels of DCLK1 mRNA (Fig. 6B), suggesting this pathway could be recruited by A-443654 to reduce α-synuclein. We performed a high-throughput screen of 155,885 compounds at multiple doses using a genome-edited cell line that expresses α-synuclein fused to luciferase under native expression control. We identified A-443654, a pan AKT inhibitor as a potent inhibitor of α-synuclein transcription that normalized the abundance of monomeric and/or oligomeric forms of α-synuclein in ATXN2-Q58 HEK-293 cells, 3XSNCA fibroblasts, and 3XSNCA iPSC-derived dopaminergic neurons. In addition, A-443654 normalized autophagy dysfunction and ER stress in ATXN2-Q58 cells. A-443654 may represent a useful scaffold for developing an effective therapeutic for PD and may also serve as a tool for further interrogating the contributions of AKT signaling on PD pathogenesis. Other efforts to silence SCNA expression include oligonucleotide-based therapeutics (antisense oligonucleotides and RNAi) and the development of small molecules lowering SNCA transcription, translation, or posttranslational half-life. Reduction of SNCA expression in PD mouse models was achieved using antisense oligonucleotide, which improve motor control and other phenotypes (32Alarcon-Aris D. Recasens A. Galofre M. Carballo-Carbajal I. Zacchi N. Ruiz-Bronchal E. Pavia-Collado R. Chica R. Ferres-Coy A. Santos M. Revilla R. Montefeltro A. Farinas I. Artigas F. Vila M. et al.Selective alpha-synuclein knockdown in monoamine neurons by intranasal oligonucleotide delivery: Potential therapy for Parkinson's disease.Mol. Ther. 2018; 26: 550-567Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 33Uehara T. Choong C.J. Nakamori M. Hayakawa H. Nishiyama K. Kasahara Y. Baba K. Nagata T. Yokota T. Tsuda H. Obika S. Mochizuki H. Amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotides targeting alpha-synuclein as a novel therapy for Parkinson's disease.Sci. Rep. 2019; 9: 7567Crossref PubMed Scopus (28) Google Scholar). Reduction of SNCA expression has also been demonstrated in PD mice using siRNAs or shRNA plasmids, and efficacy was enhanced when complexing therapeutic nucleic acids with nanoparticles (34Niu S. Zhang L.K. Zhang L. Zhuang S. Zhan X. Chen W.Y. Du S. Yin L. You R. Li C.H. Guan Y.Q. Inhibition by multifunctional magnetic nanoparticles loaded with alpha-synuclein RNAi plasmid in a Parkinson's disease model.Theranostics. 2017; 7: 344-356Crossref PubMed Scopus (70) Google Scholar, 35Helmschrodt C. Hobel S. Schoniger S. Bauer A. Bonicelli J. Gringmuth M. Fietz S.A. Aigner A. Richter A. Richter F. Polyethylenimine nanoparticle-mediated siRNA delivery to reduce alpha-synuclein expression in a model of Parkinson's disease.Mol. Ther. 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A small molecule screen of 1124 compounds identified multiple β2-adrenoreceptor agonists that lower SNCA mRNA abundance (38Mittal S. Bjornevik K. Im D.S. Flierl A. Dong X. Locascio J.J. Abo K.M. Long E. Jin M. Xu B. Xiang Y.K. Rochet J.C. Engeland A. Rizzu P. Heutink P. et al.beta2-Adrenoreceptor is a regulator of the alpha-synuclein gene driving risk of Parkinson's disease.Science. 2017; 357: 891-898Crossref PubMed Scopus (212) Google Scholar), resulting in an epidemiologic study of nearly 2 million adults that indicated that the use of β2-adrenoreceptor agonists could significantly reduce the risk of PD (39Gronich N. Abernethy D.R. Auriel E. Lavi I. Rennert G. Saliba W. beta2-Adrenoceptor agonists and antagonists and risk of Parkinson's disease.Mov. Disord. 2018; 33: 1465-1471Crossref PubMed Scopus (41) Google Scholar). Posiphen is another compound that has been proposed for PD, as it inhibits 5′-UTR-mediated SNCA translation (40Mikkilineni S. Cantuti-Castelvetri I. Cahill C.M. 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