Levodopa

Carbidopa (CD, (2S)-3-(3,4-dihydroxyphenyl)-2-hydrazinyl-2-methylpropanoic acid), levodopa (LD, (2S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid), vanillin (4-hydroxy-3-methoxybenzaldehyde), acetonitrile (MeCN), ethanol (EtOH), dimethyl sulfoxide (DMSO, methylsulfinylmethane), methanol (MeOH), and hydrogen chloride (HCl) were purchased from Sigma-Aldrich (Milan, Italy). Brand drug Sinemet (200 mg levodopa, 50 mg Carbidopa, 35 mg excipients) was obtained from MSD (Rome, Italy), and its pharmaceutical alternative (generic drug) Hexal (200 mg levodopa, 50 mg Carbidopa, 194 mg excipients) was produced by Sandoz (Basel, Switzerland). All chemicals were of analytical reagent grade and used as received without any further purification. All solutions were prepared using water obtained from Milli-Q Water Purification System (resistivity ≥ 18 MΩcm) (Germany, www.merckmillipore.com).

6-OHDA (Sigma Aldrich) was prepared at 5 mg/mL in normal saline. Levodopa was prepared (0.5 mg/mL Sigma Aldrich) with benserazide (0.25 mg/mL, Sigma Aldrich) in normal saline and always administered at 5 mg/kg.

Escherichia coli DH5a or BL21 were routinely grown aerobically in Luria-Broth (LB) at 37 °C degrees with continuous agitation. Other strains listed in Supplementary Table 6 were grown anaerobically (10% H₂, 10% CO₂, 80% N₂) in a Don Whitley Scientific DG250 Workstation (LA Biosystems, Waalwijk, The Netherlands) at 37 °C in an enriched beef broth based on SHIME medium^{37 (link)} (Supplementary Table 7). Bacteria were inoculated from −80 °C stocks and grown overnight. Before the experiment, cultures were diluted 1:100 in fresh medium from overnight cultures. Levodopa (D9628, Sigma, The Netherlands), carbidopa (C1335, Sigma), benserazide (B7283, Sigma), or methyldopa (857416, Sigma) were supplemented during the lag or stationary phase depending on the experiment. Growth was followed by measuring the optical density (OD) at 600 nM in a spectrophotometer (UV1600PC, VWR International, Leuven, Belgium).

Levodopa (sigma) and carbidopa (sigma) at 1:1 were administered orally once daily mixed in either ensure pudding or cottage cheese or marshmallows at 3–12 mg/Kg B.wt. The drug and vehicle mix was prepared fresh every day and was administered 5 days a week for two consecutive weeks. During the first week of L-Dopa therapy, PDRS and ORTBD were performed on the parkinsonian marmosets (n = 3) 1hr after the administration of vehicle or L-DOPA. Actiwatch analysis was performed during the second week of L-Dopa therapy. For the activity analysis, the actiwatch was setup to start recording at 9:00 AM and the vehicle or drugs was administered at 11:30 AM (after 2.5 hrs of baseline recording). The actiwatches were collected after 24hrs of acquisition, the data was downloaded and analyzed using the sleep analysis-7 software. The activity analysis was performed three times in a week on the three parkinsonian marmosets. The analyzed data was then exported to excel and plotted using the Graph pad prism statistical software.

Methyl-4-phenylpyridine (MPP⁺; #D048), 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP; #M0896) and levodopa (#D9628) were obtained from Sigma (St. Louis, MO, USA). An ABC reagent Box (Vector PK-6101 Rabbit IgG) and Golgi staining kit (PK401) were obtained from FD NeuroTechnologes (Columbia, MD, USA). P-tau (Ser396; #ab109390) and t-tau (#ab32057) were purchased from Abcam (Cambridge, MA), while GSK-3β (#12456) and phosphorylated GSK-3β (p-GSK-3β, ser9, #9323) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-tyrosine hydroxylase (TH) was obtained from Santa Cruz (Dallas, TX, USA). Immoblilon PVDF membranes (#ISEQ00010) and Immobilon Western Chemiluminescent HRP Substrate (#WBKLS0100) were purchased from Merck Co. (Darmstadt, Germany).
SPF grade C57BL/6 mice, male 6–7 weeks, weight 22–27 g, were purchased from Guangdong Experimental Animal Center, license number: SYXK (Yue) 2016–0167, then free drinking water, feeding to 10–11 weeks. All of the experimental protocols were approved by the Institutional Animal Care and Use Committee of Southern Medical University (Guangzhou, Guangdong, China).