Age-dependent accumulation of oligomeric SNCA/α-synuclein from impaired degradation in mutant LRRK2 knockin mouse model of Parkinson disease: role for therapeutic activation of chaperone-mediated autophagy (CMA)

Parkinson disease (PD) is an age-related neurodegenerative disorder associated with misfolded SNCA/α-synuclein accumulation in brain. Impaired catabolism of SNCA potentiates formation of its toxic oligomers. <i>LRRK2</i> (leucine-rich repeat kinase-2) mutations predispose to familial and sporadic PD. Mutant LRRK2 perturbs chaperone-mediated-autophagy (CMA) to degrade SNCA. We showed greater age-dependent accumulation of oligomeric SNCA in striatum and cortex of aged LRRK2^R1441G knockin (KI) mice, compared to age-matched wildtype (WT) by 53% and 31%, respectively. Lysosomal clustering and accumulation of CMA-specific LAMP2A and HSPA8/HSC70 proteins were observed in aged mutant striatum along with increased GAPDH (CMA substrate) by immunohistochemistry of dorsal striatum and flow cytometry of ventral midbrain cells. Using our new reporter protein clearance assay, mutant mouse embryonic fibroblasts (MEFs) expressing either SNCA or CMA recognition ‘KFERQ’-like motif conjugated with photoactivated-PAmCherry showed slower cellular clearance compared to WT by 28% and 34%, respectively. However, such difference was not observed after the ‘KFERQ’-motif was mutated. LRRK2 mutant MEFs exhibited lower lysosomal degradation than WT indicating lysosomal dysfunction. LAMP2A-knockdown reduced total lysosomal activity and clearance of ‘KFERQ’-substrate in WT but not in mutant MEFs, indicating impaired CMA in the latter. A CMA-specific activator, AR7, induced neuronal LAMP2A transcription and lysosomal activity in MEFs. AR7 also attenuated the progressive accumulation of both intracellular and extracellular SNCA oligomers in prolonged cultures of mutant cortical neurons (DIV21), indicating that oligomer accumulation can be suppressed by CMA activation. Activation of autophagic pathways to reduce aged-related accumulation of pathogenic SNCA oligomers is a viable disease-modifying therapeutic strategy for PD. <b>Abbreviations:</b> 3-MA: 3-methyladenine; AR7: 7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; CMA: chaperone-mediated autophagy; CQ: chloroquine; CSF: cerebrospinal fluid; DDM: n-dodecyl β-D-maltoside; DIV: days in vitro; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell sorting; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GWAS: genome-wide association studies; HSPA8/HSC70: heat shock protein 8; KFERQ: CMA recognition pentapeptide; KI: knockin; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LDH: lactate dehydrogenase; LRRK2: leucine-rich repeat kinase 2; MEF: mouse embryonic fibroblast; NDUFS4: NADH:ubiquinone oxidoreductase core subunit S4; NE: novel epitope; PD: Parkinson disease; RARA/RARα: retinoic acid receptor, alpha; SNCA: synuclein, alpha; TUBB3/TUJ1: tubulin, beta 3 class III; WT: wild-type

帕金森病（Parkinson disease, PD）是一种与年龄相关的神经退行性疾病，其发病与脑内错误折叠的α-突触核蛋白（SNCA/α-synuclein）聚集密切相关。SNCA的代谢受损会促进其毒性寡聚体的形成。富亮氨酸重复激酶2（leucine-rich repeat kinase-2, LRRK2）的突变会增加家族性和散发性PD的发病风险。突变型LRRK2会破坏分子伴侣介导的自噬（chaperone-mediated autophagy, CMA）对SNCA的降解过程。我们发现，与同龄野生型（wildtype, WT）小鼠相比，LRRK2^R1441G敲入（knockin, KI）小鼠的纹状体和皮层中，寡聚化SNCA的年龄依赖性聚集程度分别升高53%和31%。通过背侧纹状体免疫组化和腹侧中脑细胞流式细胞术检测，我们在老年突变型小鼠纹状体中观察到溶酶体聚集以及CMA特异性蛋白LAMP2A和HSPA8/HSC70的积累，同时CMA底物GAPDH的水平也有所升高。利用我们开发的新型报告蛋白清除实验，分别表达SNCA或与光活化PAmCherry（photoactivated-PAmCherry）偶联的CMA识别"KFERQ"样基序的突变型小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs），其细胞清除速率较WT分别低28%和34%。然而，当"KFERQ"基序发生突变后，这一差异便不复存在。LRRK2突变型MEFs的溶酶体降解能力低于WT，表明其存在溶酶体功能障碍。在WT细胞中敲低LAMP2A会降低总溶酶体活性以及"KFERQ"底物的清除能力，但在突变型MEFs中无此效应，这表明后者的CMA功能受损。一种CMA特异性激活剂AR7（7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine）可诱导神经元LAMP2A的转录并提升MEFs的溶酶体活性。AR7还可减轻突变型皮层神经元（体外培养第21天，days in vitro, DIV21）长期培养过程中细胞内和细胞外SNCA寡聚体的进行性聚集，表明CMA激活可抑制寡聚体的聚集。激活自噬通路以减少年龄相关性致病性SNCA寡聚体的聚集，是一种可行的PD疾病修饰治疗策略。

<b>缩略语对照表：</b>3-MA: 3-甲基腺嘌呤（3-methyladenine）；AR7: 7-氯-3-(4-甲基苯基)-2H-1,4-苯并恶嗪（7-chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine）；CMA: 分子伴侣介导的自噬（chaperone-mediated autophagy）；CQ: 氯喹（chloroquine）；CSF: 脑脊液（cerebrospinal fluid）；DDM: 正十二烷基β-D-麦芽糖苷（n-dodecyl β-D-maltoside）；DIV: 体外培养天数（days in vitro）；ELISA: 酶联免疫吸附试验（enzyme-linked immunosorbent assay）；FACS: 荧光激活细胞分选术（fluorescence-activated cell sorting）；GAPDH: 甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase）；GWAS: 全基因组关联研究（genome-wide association studies）；HSPA8/HSC70: 热休克蛋白8（heat shock protein 8）；KFERQ: CMA识别五肽（CMA recognition pentapeptide）；KI: 敲入（knockin）；LAMP1: 溶酶体相关膜蛋白1（lysosomal-associated membrane protein 1）；LAMP2A: 溶酶体相关膜蛋白2A（lysosomal-associated membrane protein 2A）；LDH: 乳酸脱氢酶（lactate dehydrogenase）；LRRK2: 富亮氨酸重复激酶2（leucine-rich repeat kinase 2）；MEF: 小鼠胚胎成纤维细胞（mouse embryonic fibroblast）；NDUFS4: NADH:泛醌氧化还原酶核心亚基S4（NADH:ubiquinone oxidoreductase core subunit S4）；NE: 新型表位（novel epitope）；PD: 帕金森病（Parkinson disease）；RARA/RARα: 视黄酸受体α（retinoic acid receptor, alpha）；SNCA: α-突触核蛋白（synuclein, alpha）；TUBB3/TUJ1: Ⅲ类β微管蛋白（tubulin, beta 3 class III）；WT: 野生型（wild-type）