PT109B, a Multikinase Inhibitor, Converts Astrocytes into Dopaminergic Neurons and Alleviates Parkinson's Disease in Mice

PT109B, a Multikinase Inhibitor, Converts Astrocytes into Dopaminergic Neurons and Alleviates Parkinson's Disease in Mice | 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 Research Article PT109B, a Multikinase Inhibitor, Converts Astrocytes into Dopaminergic Neurons and Alleviates Parkinson's Disease in Mice Cailv Wei, Yang Yang, Tsz Hei Fong, Yuan Liu, Shisong Wang, Chao Ding, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6428230/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Parkinson’s disease (PD) is characterized by the progressive loss of dopaminergic neurons (DANs), leading to motor dysfunction, while current treatments fail to restore neuronal loss. Reprogramming astrocytes into induced DANs by small molecules offers a promising therapeutic strategy, but existing methods face challenges including low efficiency and complex mechanisms. PT109B, a novel multi-kinase inhibitor, has demonstrated neurogenic and synaptogenic potential in neural progenitor cells, as well as glioblastoma differentiation capacity, yet its ability to directly convert astrocytes into functional DANs and its therapeutic effects in PD remain unclear. Methods Primary rat midbrain astrocytes were treated with 10 µM PT109B to evaluate reprogramming efficiency via immunofluorescence (GFAP, MAP2, NeuN, TH, DAT) and electrophysiological recordings. RNA sequencing was performed at 1.5, 3, and 6 hours post-treatment to assess transcriptional changes. In vivo, PT109B (100 mg/kg) was administered orally for 12 weeks in 6-OHDA-induced PD mice, with astrocytes labeled by AAV5-GFAP-EGFP. Behavioral tests (apomorphine rotation, pole test, rotarod, and open field), retrograde tracing, and immunohistochemistry were conducted to evaluate therapeutic effects. Results PT109B initiated astrocyte-to-neuron conversion as early as 3 hours, yielding 20% TH⁺ dopaminergic neurons by 2 weeks in vitro, with mature electrophysiological properties for action potentials, sodium currents and sustained dopamine release (> 3 months). Mechanistically, PT109B drove this conversion through cell cycle arrest, astrocytic activation, and upregulation of key basic Helix-Loop-Helix (b-HLH) transcription factors (NeuroD1, Ascl1, Ngn2). In vivo , oral administration of PT109B in a 6-OHDA-induced PD mouse model exhibited significant therapeutic efficacy by reprogramming astrocytes to functional neurons in the striatum, leading to improved motor functions. Conclusions PT109B efficiently converts astrocytes into functional induced DANs through rapid reprogramming and ameliorates PD-related pathology and motor deficits, presenting a safe and effective single-molecule therapeutic strategy for PD. Astrocytes Reprogramming Induced dopaminergic neurons PT109B Parkinson’s disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by motor dysfunction, with the pathological hallmark being the selective loss of dopaminergic neurons (DANs) in the substantia nigra pars compacta[ 1 , 2 ]. Current pharmacological treatments, including levodopa and selegiline, primarily alleviate symptoms but fail to halt disease progression or prevent neuronal loss[ 3 , 4 ]. This limitation underscores the urgent need for innovative therapeutic strategies capable of regenerating DANs to address the underlying pathology of PD. Astrocytes, the most abundant non-neuronal cell population in the brain, share a common neuroectodermal origin with neurons and exhibit shared region-specific transcriptional and epigenetic characteristics[ 5 , 6 ]. Recent advances in regenerative medicine have highlighted the potential of reprogramming resident astrocytes into neurons as a promising approach for PD treatment[ 7 , 8 ]. Gene therapy-based reprogramming, having effective in preclinical models, faces significant translational challenges, including the risk of gene mutations, genetic toxicity, and off-target effect[ 9 ]. In contrast, chemical reprogramming offers a non-genotoxic alternative with substantial clinical potential[ 9 – 11 ]. Although small molecule combinations have demonstrated the ability to transform astrocytes into neurons in the models of neurodegenerative diseases[ 12 – 16 ], this approach is hindered by several limitations, including the complexity of multi-component systems, low reprogramming efficiency, and poorly understood mechanisms[ 10 ]. Thus, identifying a single small molecule capable of efficiently reprogramming astrocytes into functional neurons will not only deepen our understanding of cell fate plasticity but also provide a promising approach for PD therapeutics. We unexpectedly discovered that PT109B, a multi-kinase inhibitor, promotes neurogenesis in C17.2 cells and enhances synaptogenesis in primary cultured rat hippocampal neurons, and it also induces glioblastoma differentiation via the PTBP1/PKM1/PKM2 pathway[ 17 , 18 ]. In addition to its biological activity, PT109B demonstrates favorable pharmacokinetic properties, including a half-life of 3.14 ± 0.68 hours and efficient blood-brain barrier penetration[ 19 ], positioning it as a promising therapeutic candidate for central neurodegenerative diseases. In this study, we developed a streamlined protocol using PT109B as a single-agent inducer to rapidly and directly reprogram astrocytes into functional induced dopaminergic neurons (iDANs). Remarkably, in vivo application of PT109B not only alleviated motor deficits in a 6-hydroxydopamine (6-OHDA)-induced PD mouse model, but also increased the number of DANs in the striatum and substantia nigra. Our findings identify that PT109B as a novel small molecule with the capacity to efficiently reprogram astrocytes into iDANs, presenting a substitute approach for the treatment of neurodegenerative disorders. Materials and methods Animal 8-week-old male C57BL/6 mice were obtained from Zhuhai BesTest Bio-Tech Co. and used to evaluate the anti-PD effects of PT109B. All animals were housed in a specific pathogen-free facility maintained at a controlled temperature of 21 ± 1℃ and relative humidity of 65%, under a 12 hours light/dark cycle. Mice were acclimatized for at least 1 week prior to experiments. All experimental procedures were conducted in accordance with the ethical guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University. Preparation of medication PT109B was prepared as an oral suspension for administration via gavage. Briefly, PT109B was dissolved in physiological saline containing 20% (w/v) hydroxypropyl-β-cyclodextrin (HP-β-CD) to achieve a final concentration of 20 mg/mL. The suspension was freshly prepared and vortexed thoroughly to ensure homogeneity. PD mice models were induced by 6-OHDA prior to treatment. For dosing, mice received 100 mg/kg of PT109B or 10 mg/kg Selegiline administered orally via gavage once daily for a total duration of 3 months. Establishment of the 6-OHDA-induced PD mice model The 6-OHDA-induced PD mice model was established according to referenced protocols[ 20 ]. Briefly, mice were anesthetized using an isoflurane anesthesia system and securely positioned on a stereotaxic apparatus to stabilize the head. After shaving the surgical site and disinfecting with alcohol or iodine, a midline incision was made on the scalp, and the periosteum was gently removed to expose the skull and achieve hemostasis. The surgical area was further cleaned with 3% hydrogen peroxide, and the bregma point was identified as the anatomical reference. Using the stereotaxic atlas for mice, the coordinates for the mFB were determined as follows: anterior-posterior (AP): -1.2 mm, medio-lateral (ML): +1.3 mm, and dorso-ventral (DV): -4.8 mm. A small burr hole was drilled at the target site, and 6-OHDA solution (4 µg in 2 µL) was injected into the right mFB area using a 10 µL microsyringe at a rate of 0.5 µL/min under dim lighting conditions. The needle was retained for 5 minutes post-injection to minimize backflow and withdrawn slowly over 3 minutes. Successful lesioning was validated by observing contralateral circling behavior, a hallmark of unilateral dopaminergic depletion. Three weeks post-lesioning, mice were screened for successful modeling using a unilateral rotation test induced by subcutaneous administration of apomorphine (0.5 mg/kg). Mice exhibiting > 150 contralateral rotations within 30 minutes were considered to have successfully modeled PD and were included in subsequent experiments. AAV5-GFAP-EGFP labeling of astrocytes in the mouse brain Astrocytes were labeled in the mouse brain using AAV5-GFAP-EGFP (Shanghai Genechem Co.,Ltd). Mice were anesthetized and securely positioned in a stereotaxic frame. The microinjector was aligned to target the striatum (coordinates: AP: +1.0 mm, ML: +1.6 mm, DV: -2.8 mm) and substantia nigra (coordinates: AP: -3.0 mm, ML: +1.3 mm, DV: -4.35 mm). After stabilizing the injector for 5 minutes to ensure precision, AAV5-GFAP-EGFP (1×10 12 vg/mL) was injected into the cortex at a constant rate of 0.2 µL/min for 5 minutes. To minimize reflux, the injector was retained in place for an additional 5 minutes before being slowly withdrawn. The viral construct was sourced from Jikai Gene. Retrograde labeling of dopaminergic projections using Cholera toxin B (CTB) Retrograde tracing of dopaminergic neuronal projections from the striatum to the substantia nigra was performed using CTB, conjugated to Alexa Fluor™ 488, as previously described[ 15 ]. Mice were anesthetized and mounted on a stereotaxic frame, with the microinjector aligned to the striatum (coordinates: AP: +1.0 mm, ML: +1.6 mm, DV: -2.8 mm). After stabilizing the injector for 5 minutes, CTB (10 mg/mL) was delivered at a constant rate of 0.2 µL/min for 5 minutes. To prevent tracer reflux, the injector was held in place for an additional 5 minutes before being withdrawn slowly. Apomorphine-induced rotation test Apomorphine-induced rotation was performed 3 weeks after 6-OHDA lesion and 15 weeks after PT109B or selegiline treatment. Briefly, 10 minutes after intraperitoneal injection of apomorphine (5 mg/kg dissolved in ice-cold saline solution), each mouse was placed in a circular basin, and contralateral rotations were recorded over a 30-minute period. Open field test The open field test was conducted to evaluate spontaneous locomotor activity, exploratory behavior, and anxiety levels in a novel environment. The apparatus consisted of a square arena measuring 40 × 40 × 40 cm for mice. Prior to testing, animals were acclimated to the experimental room for 2 hours under consistent lighting and noise conditions to minimize stress. Each animal was gently placed in the center of the arena and allowed to explore freely for 5 minutes. Locomotor parameters, including total distance traveled, time spent in the center zone, distance traveled in the center, and movement speed, were automatically recorded using tracking software. To reduce inter-animal interference, tested individuals were temporarily housed in separate cages. After each session, the arena was thoroughly cleaned with 75% ethanol to eliminate odor cues and allowed to dry before subsequent trials. Pole test The pole test was performed as previously described to assess motor coordination and agility in mice[ 21 ]. The apparatus consisted of an 80 cm tall vertical pole (3 cm in diameter) with a circular platform (12 cm diameter) mounted at the top. The platform surface was covered with hemp cloth to provide traction. Each mouse was placed head-down on the platform, and the time taken to descend to the base of the pole was recorded. The test was repeated three times per mouse, and the average descent time was calculated for analysis. Hindlimb clasping test Motor impairment was assessed using the hindlimb clasping Test. Mice were gently suspended by the tail, and hind limb movements were scored over a 15-second interval as follows: 0 = both hind limbs fully extended (abducted); 1 = partial extension of both hind limbs; 2 = partial flexion (adduction) of both hind limbs; 3 = both hind limbs fully flexed. All tests were video-recorded to ensure accurate scoring and subsequent review. Rotarod test Motor coordination and endurance were evaluated using the rotarod test. Mice underwent a 1-day acclimatization training session on the Rotarod apparatus at constant speeds of 10, 15, and 20 rpm for 10 minutes each. During testing, the Rotarod speed was set to an optimal baseline (e.g., 15 rpm), and the latency to fall was recorded over three trials. The average latency across trials was used for statistical analysis. Whole-cell patch clamp for electrophysiological activity Whole-cell patch-clamp recordings were performed on cultured astrocytes/iDANs to assess electrophysiological activity. Artificial Cerebrospinal Fluid (ACSF) was used for cell incubation contained (in mM) 122 NaCl, 2.5 KCl, 1.2 NaH 2 PO 4 , 24 NaHCO 3 , 12.5 D-glucose, 2 CaCl 2 , 1 MgSO 4 , 1 MgCl 2 , and 5 HEPES. Pipette internal solution (in mM): 128 K-gluconate,10 NaCl, 2 MgCl 2 , 0.5 EGTA, 10 HEPES, 0.4 Na 2 GTP, and 4 Na 2 ATP. PT109B-induced astrocytes cultured on coverslips were incubated in oxygenated ACSF (95% O₂ and 5% CO₂) for 30 minutes prior to recording. Cells were then transferred to a recording chamber continuously perfused with oxygenated ACSF at room temperature (25℃-30℃). Electrophysiological Recordings from Brain Slices: Electrophysiological recordings were performed on brain slices to assess neuronal activity. Experimental mice were anesthetized with 1% sodium pentobarbital (intraperitoneal injection) and decapitated. The brain was rapidly removed and immersed in ice-cold, oxygenated slicing solution (95% O 2 and 5% CO 2 ) at 0℃. After 1 minute of cooling, the brain was trimmed and mounted on a vibratome stage (Leica VT1200). Brain slices containing the target region were cut at a thickness of 300 µm at a speed of 0.09 mm/s. Slices were transferred to a chamber containing oxygenated ACSF and incubated at 35°C for 30 minutes, followed by an additional 60 minutes at room temperature. The slicing solution was prepared as follows (in mM): 110 choline chloride, 7 MgSO 4 , 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 25 D-glucose, 11.6 sodium L-ascorbate, 3.1 sodium pyruvate, and 0.5 CaCl 2 . The ACSF for slice incubation contained (in mM) 127 NaCl, 2.5 KCl, 1.25 NaH 2 PO 4 , 25 NaHCO 3 , 25 D-glucose, 2 CaCl 2 , and 1 MgCl 2 . The internal potassium-based pipette solution used is the same with cultured cells recordings. Whole-cell patch-clamp recordings were performed using glass pipettes (5–8 MΩ) filled with the potasium-based internal solution, and cells were recorded utilizing a HEKA EPC10 dual patch-clamp amplifier (HEKA Elektronik, Germany). Signals were acquired at a sampling rate of 10 kHz and filtered at 2 kHz. For voltage-clamp experiments, cells were held at a holding potential of -70 mV. Recorded data were analyzed offline using Clampfit 11 software (Molecular Devices, USA). Single-molecule induced reprogramming of astrocytes into neurons The neuronal induction medius was prepared by combining 50 mL of Neurobasal medium with key supplements: 1% N2 (Gibco, 17502001), 1% B27 (Gibco, A3582801), 1% Glutamax (Gibco, 35050061), and 1% penicillin/streptomycin. To this base medium, the small molecule PT109B (10 µM) and neurotrophic factors—BDNF (PeproTech, 450-02-50UG), GDNF (PeproTech, 450-10-10UG), b-FGF (PeproTech, 100-18B-50UG)—were added to generate the PT109B-containing neuronal induction medium. For reprogramming, mature astrocytes were initially cultured for 1–3 days in a 1:1 mixture of the PT109B-containing neuronal induction medium and high-glucose DMEM. After this transitional phase, the medium was fully replaced with the PT109B-containing neuronal induction medium. astrocytes were maintained in this medium for 14–90 days, with daily monitoring of cell morphology and viability. The medium was refreshed every 2–3 days, with the frequency adjusted based on cellular health and metabolic activity. When whole-cell patch clamp was performed to detect electrophysiological function, T3 (Bioss, bs-0339P, 50 µM) was added to neuron induction medium. RNA-seq sequencing Total RNA extracted using TRIzol was submitted to Genedenovo Biotechnology Co., Ltd. (Guangzhou, China) for RNA sequencing. RNA integrity was verified using an Agilent Bioanalyzer, and libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (Illumina). Sequencing was performed on an Illumina NovaSeq 6000 platform, generating paired-end 150 bp reads. Omics data analysis Raw RNA-seq data were processed and analyzed on the Genedenovo multi-omics data mining system platform. Following quality control and read alignment to the reference genome, differentially expressed genes (DEGs) were identified using DESeq2. Gene ontology (GO) enrichment analysis was performed using the DAVID platform ( https://david.ncifcrf.gov/ ). Protein-protein interaction networks were constructed using the STRING database ( https://string-db.org/ ) and visualized using Cytoscape software (version 3.8.2). Core network modules were identified and analyzed using the MCODE plugin in Cytoscape. Cell culture Culture of primary midbrain astrocytes from newborn SD rats astrocytes were isolated from the midbrains of 1–3 days old Sprague-Dawley (SD) rats. Brains were harvested and placed in precooled D-HANKS buffer. Under a microscope, the meninges and blood vessels were meticulously removed to isolate the midbrain, which was then transferred to pre-cooled serum-free DMEM medium. Tissue digestion was performed using 0.125% trypsin for 15 minutes at 37℃, and the reaction was quenched by adding an equal volume of serum-containing medium. After centrifugation at 1,000 rpm for 8 minutes, the supernatant was discarded. The cell pellet was resuspended in serum-containing medium and seeded at a density of 2.0×10 5 cells/cm² in astrocytes medium (89% DMEM, 10% FBS, 1% penicillin/streptomycin). The medium was refreshed every three days. On day 7, cultures were subjected to orbital shaking at 37°C and 200 rpm for 12 hours to remove contaminating microglia and oligodendrocytes. The purified astrocytes were then separated using 0.25% trypsin-EDTA and seeded onto poly-L-lysine-coated (0.01 mg/mL) culture plates for subsequent experiments. Culture of primary cortical neurons from newborn SD rats Primary cortical neurons were isolated from 0–3 days old SD rat pups. The cortical tissues were excised and minced into approximately 1 mm³ pieces using sterile ophthalmic scissors. The minced tissue was enzymatically digested in 0.125% trypsin and DNase (8 mg/mL) at 37°C for 15 minutes with gentle agitation every 5 minutes. Digestion was halted by adding complete medium. The mixture was centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded. The pellet was resuspended in DMEM supplemented with DNase, gently pipetted, and briefly centrifuged at 1,500 rpm to remove cell clumps and debris. The resulting supernatant was centrifuged again at 1,000 rpm for 5 minutes, and the pellet was resuspended in 10% FBS-DMEM. Neurons were counted and seeded at 2–5 × 10 4 cells/cm² onto poly-L-lysine-coated (0.01 mg/mL) dishes or plates. After 2–4 hours of incubation for cell adherence, the medium was replaced with neurobasal medium supplemented with 1% B27, 0.25 mM glutamine, and 100 U/mL penicillin/streptomycin. Primary neurons were identified on day 3, and experiments commenced on day 7. Immunofluorescence Cell immunofluorescence staining : Cells were washed three times with phosphate-buffered saline (PBS) to remove residual serum and cellular debris. After washing, cells were fixed with 4% (w/v) paraformaldehyde in PBS for 30 minutes at room temperature, and permeabilized with 0.4% Triton X-100 in PBS for 15 minutes to allow antibody access to intracellular targets. Non-specific binding sites were blocked by incubating cells with 10% normal goat serum in PBS for 1 hour at room temperature. Primary antibodies were diluted in blocking solution and applied to cells overnight at 4℃. The next day, cells were washed three times with PBS to remove unbound primary antibodies and incubated with species-specific Alexa Fluor-conjugated secondary antibodies (1:2000) for 1 hour at room temperature. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime, C1002) for 10 minutes. Immunofluorescence staining of brain tissue sections : Brains were isolated and post-fixed in 4% (w/v) paraformaldehyde overnight at 4℃, followed by cryoprotection in 30% sucrose in PBS for 72 hours at 4℃. Coronal brain sections (20 µm thick) were collected using a sliding microtome (Leica). Sections were blocked with 10% normal goat serum in PBS for 1 hour at room temperature and incubated with primary antibodies overnight at 4℃. Primary antibodies included GFP (Rabbit; 1:200; Invitrogen), NeuN (Mouse; 1:200; Abcam), GFAP (Mouse; 1:200; Invitrogen) and MAP2 (Mouse; 1:200; Abcam). After washing three times with PBS, sections were incubated with corresponding Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (Invitrogen; 1:1000) for 1 hour at room temperature. Images were captured using a Zeiss LSM700 or Nikon A1R confocal microscope for analysis. Detection of dopamine (DA) in cell culture medium DA levels in the supernatant were quantified using a competitive ELISA kit (Elabscience, EEL144) following the manufacturer’s protocol. Upon completion of the 28–30 days culture period of PT109B-induced cells, the culture medium was collected and centrifuged at 1000 × g for 20 minutes at 4°C to remove cell debris and impurities. Briefly, a 96-well plate was pre-coated with dopamine antigens. DA present in the samples or standards competed with the immobilized antigens for binding sites on biotin-conjugated anti-dopamine antibodies. Unbound components were removed through washing steps. Horseradish peroxidase-labeled avidin was added, which binds specifically to the biotin-labeled antibodies, forming an immune complex. After another wash step, the substrate 3,3’,5,5’-tetramethylbenzidine was added. 3,3’,5,5’-tetramethylbenzidine was enzymatically converted to a blue product by Horseradish peroxidase, which turned yellow upon addition of the stop solution (1 M sulfuric acid). Optical density was measured at 450 nm using a microplate reader. The concentration of DA in the samples was inversely proportional to the OD450 values and was determined by interpolation from a standard curve generated using known concentrations of DA. BrdU method for cell proliferation detection A BrdU working solution (10 µM) was prepared using freshly pre-warmed cell culture medium. Cells were subsequently treated with neuronal induction medium supplemented with PT109B (10 µM) for 3 or 7 days. The BrdU working solution was added to cultured cells and incubated at 37℃ for 4 hours to allow BrdU incorporation into proliferating cells’ DNA. Following incorporation, the BrdU-containing medium was replaced with normal culture medium, and cells were further cultured for 24 hours. Cells were washed briefly with PBS and fixed in chilled 70% ethanol for 1 minute, followed by fixation in 4% paraformaldehyde at room temperature for 5 minutes. The fixative was removed, and cells were washed three times with PBS (5 minutes each). To denature DNA and expose BrdU epitopes, cells were treated with 1.5 M HCl at room temperature for 30 minutes. After HCl treatment, cells were washed twice with PBS (5 minutes each). BrdU incorporation was detected using standard immunofluorescence methods. Primary antibodies against BrdU were applied, followed by species-specific secondary antibodies conjugated to fluorescent dyes. Nuclei were counterstained with DAPI, and images were acquired using Nikon fluorescence microscope (Nikon, Eclipse Ti2-E). Western blot Cells were lysed using RIPA lysis buffer (containing protease inhibitors), and the resulting lysates were centrifuged to remove cellular debris. Protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (ThermoFisher, 23225) following the manufacturer’s instructions. Based on protein concentration quantified by BCA, 30 µg of protein per well was loaded onto the gel. Electrophoresis was performed at a constant voltage of 80–120V. The membrane was blocked with 5% bovine serum albumin or 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 2–3 hours at room temperature to prevent non-specific binding. The blocked membrane was incubated with the primary antibody at 4°C overnight and secondary antibody at room temperature for 1 hour. Protein bands were visualized using a cold charge-coupled device imaging system after exposure to enhanced chemiluminescence substrate. Cell transfection experiment 293T cells or primary astrocytes were seeded at a density of 1×10 5 cells per well in six-well plates pre-coated with poly-L-lysine. Transfection was performed using Lipofectamine 2000 (11668500, Invitrogen) according to the manufacturer’s protocol. Briefly, plasmid DNA (1 µg) or siRNA (50 nM, Table S1 ) was diluted in 200 µL of DMEM (Solution A), while 10 µL of Lipofectamine 2000 was diluted in another 200 µL of DMEM (Solution B). Solutions A and B were gently mixed and incubated at room temperature for 10 minutes. Subsequently, Solution B was added to Solution A, gently mixed, and incubated for an additional 20 minutes at room temperature to allow complex formation. The transfection reagent mixture was added dropwise to each well, and cells were incubated 24 hours at 37°C in a 5% CO₂ atmosphere. And the transfection medium was replaced with complete culture medium, and cells were cultured for an additional 24 hours. Following this, the medium was replaced with either regular medium or neuronal induction medium supplemented with PT109B (10 µM) for another 24 hours. Cell morphology was assessed under a phase-contrast microscope to monitor transfection efficiency and overall cell health. Cells were then harvested, and protein lysates were collected for subsequent biomarker analysis using Western blotting. Fluorescent quantitative PCR reaction Total RNA was extracted from cells using TRIzol reagent (Invitrogen, 15596018CN) following the manufacturer’s instructions. RNA concentration and purity were assessed using a spectrophotometer. Subsequently, cDNA was synthesized from 1 µg of total RNA using the HiFiScript gDNA Removal cDNA Synthesis Kit (CWBIO, CW2582M), following the provided protocol. qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Q121) on a StepOne Plus Real-Time PCR System (Applied Biosystems). Primer sequences are shown in Table S2 . The mRNA expression of target genes was normalized to GAPDH and calculated using the 2 -△△Ct method. Antibodies GFAP (ab68428), S100β (ab52642), NeuN (ab104224), MAP2 (ab254143), Tyrosine Hydroxylase (TH) (ab75875), Dopamine Transporter (DAT) (ab184451), Ascl1 (ab74065), Brdu (ab1893), OCT4 (ab271937), SOX2 (ab79351), Synaptophysin (ab32127), C3 (ab200999), Dlx2 (ab272902), Islet 1 (lsl1) (ab178400), Lhx8 (ab137036), DDDDK tag (Binds to FLAG tag sequence) (ab205606), Anti-Rabbit IgG (H + L) Antibody, Alexa Fluor 488 (ab150077), Anti-Mouse IgG (H + L) Antibody, Alexa Fluor 594 (ab150116), Anti-Rabbit IgG (H + L) Antibody, Alexa Fluor 647 (ab150079), Anti-Mouse IgG (H + L) Antibody, Alexa Fluor 488 (ab150113) were obtained from Abcam.Iba-1 (019–19741) was obtained from Wako.MAP2 (4542S), DCX (4604s), Ki67 (9129S), NeuroD1 (7019S), Ngn2 (13144S), Tuj1 (5568S), COX2 (12282s), c-Myc (13987S), Brn2 (12137s) were obtained from Cell Signaling Technology.MOG (A5353), Synap1 (A6344), PTBP1 (A6107), TGF-β1 (A25313) were obtained from Abclonal.VGluT1 (48-2400), GFP (A-11122), GAD67 (PA5 21397), Cholera toxin subunit B (recombinant), Alexa Fluor™ 488 (C34775), Anti-Sheep IgG (H + L) Antibody, Alexa Fluor 568 (A21099) were obtained from Invitrogen.Nurr1 (10975-2), PKM1 (15821-1-AP), REST (22242-1- AP) were obtained from Proteintech.NaV (ASC-003) was obtained from Alomone.GFAP (MAB360) was obtained from Milipore.GAPDH (AC001) was obtained from ABclonal.Flag (F3165) and HA (H9658) antibodies were obtained from Sigma - Aldrich. Statistical analysis Statistical significance was determined by Student’s t test (for two groups) or one-way or two-way ANOVA, followed by Tukey’s multiple comparisons test in using GraphPad Prism 10.4. P < 0.05 was considered significant (indicated by an asterisk in the figures). All data were reported as the mean values ± SEM. Results PT109B rapidly reprograms astrocytes into induced neurons To examine the impact of PT109B on astrocytes into neuron conversion, we systematically evaluated the temporal dynamics of PT109B mediated astrocytes fate modulation derived from midbrain (Fig. 1 a). Initial characterization confirmed the high purity (>95%) of the primary rat midbrain astrocytes cultures, which exhibited robust immunopositivity for astrocytic markers (GFAP, S100β) while lacking neuronal (NeuN, DCX), microglial (Iba1), and oligodendroglial (MOG) contaminants (Fig. S1 a-c). Firstly, following PT109B treatment (10 µM), we observed a striking temporal progression of cellular transformation. Within 24 hours, astrocytes exhibited significant morphological alterations characterized by reduced cell body size and enhanced process extension, and these changes were much more evident with longer exposure to PT109B (Fig. 1 b-f). By 7 days post-treatment of PT109B, we detected substantial molecular change, evidenced by decreased GFAP expression accompanied by upregulation of mature neuronal markers (MAP2, NeuN) and dopaminergic-specific proteins (TH, DAT), in time dependent manner (Fig. 1 g, h). Moreover, the augmented reactivity of activated astrocytes plays a pivotal role in driving their transformation into neurons. 15 PT109B triggered a pattern of astrocytic activation, with immediate (24 hours) upregulation of COX2, TNF-α, and TGF-β1, followed by sustained augmentation of C3, TNF-α, and TGF-β1 levels at 7 days (Fig. 1 i, j). In the aforementioned results, we observed that PT109B induced astrocytes activation and enhanced the levels of neuron markers as early as 1 day, suggesting that the reprogramming effects of PT109B might be initiated within 24 hours. We further examined that astrocytes reprogramming could be initiated after treatment with PT109B for 0, 0.75, 1.5, 3, 6, 12, 24, and 72 hours (Fig. 1 a). We observed that PT109B initiates cellular transformation even earlier, with significant morphological changes occurred within 0.75 hour of treatment and becoming more neuron-like over time (Fig. 1 k). Notably, after 3 hours of PT109B treatment, the protein expression level of Tuj1, an immature neuron marker, exhibited significant differences. As the PT109B treatment duration extended, the expressions of both Tuj1 and DCX were significantly elevated (Fig. 1 l, m), and the number of Tuj1 + cells was significantly higher (Fig. 1 n, o). Together, these results indicate that PT109B is a remarkably rapid and potent inducer of astrocytes-to-neuron reprogramming. PT109B reprograms midbrain astrocytes into functional iDANs Given that PT109B can rapidly reprogram astrocytes into induced neurons expressing with DAN-specific markers, we further evaluated its potential to generate mature and functional iDANs. After PT109B treatment for half a month, we observed that the emergence of MAP2 + /NeuN + cells (Fig. 2 a) and TH + /NeuN + cells (Fig. 2 b) from the astrocyte population, with these cells exhibiting neuronal morphology. Quantitatively, PT109B treatment resulted in approximately 70–85% MAP2 + and NeuN + cells (Fig. 2 a), with 20% of them expressing tyrosine hydroxylase (TH), a hallmark of dopaminergic identity (Fig. 2 b). When the PT109B treatment was extended to 1 month, the PT109B-induced cells also demonstrated a significant expression of another dopaminergic markers DAT and Nurr1 (Fig. 2 c, d), and synaptic protein Synap1 (Fig. 2 e). And a significant increase in NaV + cells was observed in astrocytes, with NaV channel signals primarily localized to the cell bodies (Fig. 2 f). Furthermore, we observed significantly elevated dopamine levels in the medium cells (Fig. 2 i), collectively confirming the acquisition of dopaminergic neuron properties. To confirm the electrophysiological maturity, we started to test the capability of action potential generation from the 1-month PT109B-treated cells. The PT109B-treated cells not only progressively acquired morphological characteristics of neurons from 1- to 3-month treatment (Fig. 2 g). The 1 to 3-month post-PT109B treatment cells displayed a rudimentary depolarized peak (half-width > 5 ms, amplitude > 50 mV) with a significant reduction in capacitance and augmentation in peak amplitude, accounting for more than 40% of recorded neuron-like cells (Fig. 2 h, j-l). However, these cells did not demonstrate a well-developed inward current, and no typical action potential (AP) was observed (Fig. 2 h). To further enhance iDANs electrophysiological functions, we supplemented PT109B with 50 nM thyroid hormone T3 for half a month. T3 plays an important role in regulating neuronal excitability and enhancing synaptic efficacy through its action on Na-K-ATPase activity, which modulates ion homeostasis and membrane potential[ 22 ]. The combination of treatment yielded more than 45% of recorded neuron-like cells exhibiting AP, particularly 15 cells generating multiple APs within 1 month (Fig. 2 m). PT109B-treated cells, especially with T3, showed significantly increased input resistance along with mature neuronal morphological and electrophysiological properties, including fine and elongated dendrite/axon-like processes, capability for continuous firing, voltage-dependent sodium and potassium currents, and spontaneous postsynaptic currents (Fig. 2 n-r). These induced properties indicate the enhanced neuronal functions and potential of neuronal network formation. Collectively, these results demonstrate that PT109B alone suffices to generate iDANs with basic structural and functional properties, while its combination with T3 produces relatively mature electrophysiological functions. PT109B directly reprograms astrocytes into iDANs without involving a neural progenitor stage It is reported that pluripotent stem cells could be generated by chemical reprogramming. The potential tumor risks of pluripotent stem cell may hamper their usage[ 23 , 24 ]. Thus, direct lineage reprogramming astrocytes by small molecular combinations provides a valuable approach for generating neurons. To address whether the reprogramming process by PT109B involving a stem cell-like intermediate stage, we systematically analyzed the expression of neural stem cell markers (SOX2, OCT4) and an immature neuronal marker (Tuj1) in astrocytes, treated with PT109B, for 3 and 7 days (Fig. S2 a). Strikingly, we observed a significant increase in the number of Tuj1 + cells in PT109B-treated astrocytes compared to the control group, whereas SOX2 + and OCT4 + cells were entirely absent (Fig. S2 b-d). Given the robust proliferative capacity of neural stem cells, we also evaluated cell proliferation during PT109B reprogramming. Astrocytes were pre-labeled with BrdU before PT109B treatment, and the presence of BrdU + and Ki67 + cells was assessed on days 3 and 7 post-treatment. Notably, the number of BrdU + and Ki67 + cells in PT109B-treated groups was significantly reduced compared to the control (Fig. S2 e-g). These findings collectively confirm that PT109B-mediated reprogramming bypasses a stem cell-like stage and concurrently suppresses cell proliferation. Further supporting this notion, we observed progressive morphological changes in astrocytes being treated with PT109B over 3, 7, and 14 days. The treated cells exhibited neuronal characteristics, including smaller cell bodies and increased length and branching of processes. Concurrently, the number of GFAP + astrocytes decreased, while the cells co-expressing GFAP, MAP2, and TH markers emerged (Fig. S2 h). Statistical analysis revealed an inverse correlation between the number of GFAP + cells and the duration of PT109B treatment, whereas the proportions of MAP2 + and TH + cells positively correlated with the treatment duration (Fig. S2 i-k). Taken together, these results suggest that PT109B directly reprograms midbrain astrocytes into functional iDANs without transitioning through a stem cell-like intermediate. PT109B reprograms astrocytes into neurons by altering multiple transcription factors Basic helix-loop-helix (bHLH) factors, such as Ascl1 (also called Mash1), NeuroD1, and Neurogenin 2 (Ngn2), play a key role in regulating the fate determination and reprogramming of astrocytes into neurons[ 25 , 26 ]. Previous studies have demonstrated that overexpression of transcription factors Ascl1 and NeuroD1 can reprogram astrocytes into iDANs[ 27 ]. To elucidate the underlying molecular mechanisms of PT109B-mediated reprogramming, we employed fluorescent quantitative PCR to analyze the mRNA expression levels of key neuronal markers and transcription factors in astrocytes following 3 days of PT109B treatment. Notably, PT109B significantly upregulated the mRNA levels of neuronal markers, including MAP2, NeuN, Tuj1, and DCX (Fig. 3 a), as well as transcription factors such as NeuroD1, Ngn2, Ascl1, Nurr1, and Dlx2 (Fig. 3 b). Furthermore, PT109B also enhanced the mRNA expressions of MAP2, TH and NeuroD1 in astrocytes following 7 days of PT109B treatment (Fig. 3 c). Then, we exposed astrocytes to PT109B for varying durations (0, 3, 6, 12, 24, 72, and 168 hours) and analyzed the expression of key bHLH transcription factor. Consistent with our previous observations, the mature neuronal marker MAP2 began to increase after 3 days of PT109B treatment (Fig. 3 d, e). Notably, within 12 hours of exposure, we observed a significant upregulation of Ascl1, Ngn2, and NeuroD1 (Fig. 3 d, e). Concurrently, levels of c-Myc, PTBP1, and REST, known inhibitors of neuronal differentiation, were markedly reduced (Fig. 3 f, g). In contrast, transcription factors associated with stem cell maintenance (Isl-1, SOX2, OCT4, and Brn2) and cholinergic neuronal differentiation (Lhx8) were largely unaffected (Fig. 3 f, g). These results suggest that PT109B specifically targets a subset of transcription factors critical for neuronal reprogramming, including c-Myc, Ascl1, Ngn2, Nurr1, NeuroD1, and Dlx2. Previous studies have shown that suppression of PTBP1 enhances the expression of NeuroD1, Ascl1, and neuronal-related genes in astrocytes[ 28 ]. Interestingly, our results revealed that PT109B significantly reduces PTBP1 levels (Fig. S3 a, b), implicating PTBP1 as a potential mediator of PT109B-induced reprogramming. To further explore this, we compared the effects of PT109B treatment and PTBP1 knockdown in astrocytes. While both interventions reduced PTBP1 levels, only PT109B induced significant morphological changes characteristic of neuronal reprogramming (Fig. S3 c, d). Moreover, even under conditions of PTBP1 overexpression, PT109B successfully reduced PTBP1 level and initiated morphological transformation (Fig. S3 e, f). These findings demonstrate that PT109B orchestrates astrocytes-to-dopaminergic neuron conversion through the coordinated regulation of bHLH transcription factors. Transcriptomic changes during PT109B initiation of astrocytes reprogramming Building on our earlier discovery that PT109B initiates astrocytes reprogramming within 3–6 hours, we sought to unravel the underlying molecular mechanisms driving this process. We conducted transcriptomic analysis at 1.5, 3, and 6 hours post-PT109B treatment. Principal component analysis (PCA) revealed a progressive divergence between PT109B-treated and control groups, indicating substantial transcriptional rewiring over time (Fig. 4 a, b). Differential gene expression analysis identified a significant number of genes with fold changes > 2 and P < 0.05 in each group. Specifically, PT109B treatment resulted in the upregulation of 647 genes and downregulation of 1179 genes at 1.5 hours, 557 upregulated and 2129 downregulated genes at 3 hours, and 707 upregulated and 1913 downregulated genes at 6 hours (Fig. 4 c). Functional annotation of these differentially expressed genes (DEGs) revealed a marked decline in pathways associated with cell cycle, RNA splicing, DNA binding, and glial cell proliferation and differentiation. Conversely, genes involved in neuronal differentiation, neurogenesis, dopamine neuron differentiation, and extracellular matrix organization exhibited gradual upregulation over time (Fig. 4 d). KEGG pathway analysis further highlighted significant enrichment in cell cycle regulation, TNF inflammatory signaling, TGF-β signaling, and neural regeneration pathways (Fig. 4 e). These findings suggest that PT109B-mediated reprogramming rapidly inhibits cell cycle progression and activates inflammation-related pathways, while simultaneously promoting neurogenesis-associated gene expressions. To identify key regulatory genes driving these early transcriptional changes, we analyzed DEGs across time points and visualized them in volcano plots, selecting the top ten significantly altered genes for further investigation (Fig. 4 f-h). Pathway impact analysis revealed that these genes primarily suppressed cell cycle and tumor-associated microRNA regulatory pathways while enhancing MAPK and TGF-β signaling (Fig. 4 i). The upregulated DEGs were predominantly associated with neurodegeneration, inflammation, DNA binding, and cytoskeletal regulation, while the downregulated DEGs were enriched in cell cycle progression, proliferation, and lipid metabolism (Fig. 4 j). Gene network analysis within these pathways identified critical roles for transcription factor-associated proteins, including Smad3, Fosb, Sp1, and Myc, whereas PTBP1 and PTBP2 showed limited interactions (Fig. 4 k). To validate the RNA sequencing results, we employed qPCR to evaluate the expression profiles of critical signaling pathways following 6 hours of PT109B-induced reprogramming. The mRNA levels of genes involved in the TGF-β/BMP signaling pathway (TGF-β1, BMP1, SMAD3), the MAPK signaling pathway (MAPK1, Fosb, Myc, Ptgs2) were significantly upregulated (Fig. S4a, b). In contrast, the mRNA levels of cell cycle regulation (Mki67, Pten) and PTBP1 were markedly downregulated (Fig. S4a-c). These results collectively demonstrate that PT109B orchestrates early transcriptional reprogramming by modulating DNA- and RNA-related functional proteins, leading to the activation of MAPK and TGF-β signaling pathways. PT109B improves motor function in 6-OHDA-induced PD mice To evaluate PT109B efficacy in vivo within the context of PD, we employed a 6-OHDA-induced PD mouse model, and selectively targeting astrocytes via adeno-associated virus (AAV5-GFAP-EGFP) delivery to the substantia nigra and striatum (Fig. S5a). Three weeks following 6-OHDA injection into the medial forebrain bundle (MFB), apomorphine-induced rotation tests confirmed successful PD modeling (Fig. S5b). Immunofluorescence analysis revealed a marked reduction in TH + neurons in the substantia nigra and striatum of PD mice compared to controls, validating the model’s neuropathological fidelity (Fig. S5c). Further, GFP + cells were found to co-localize with GFAP but not NeuN, confirming specific targeting of astrocytes rather than neurons (Fig. S5d, e). To assess the therapeutic potential of PT109B, PD mice were orally administered PT109B (100 mg/kg) for 12 weeks. At week 18, retrograde labeling was achieved via cholera toxin B (CTB) injection into the striatum, followed by behavioral and pathological assessments 1 week later (Fig. 5 a). Apomorphine-induced rotation tests demonstrated a significant reduction in rotational behavior in PT109B-treated PD mice compared to untreated PD controls, while selegiline (a standard PD treatment) showed only a moderate, non-significant effect (Fig. 5 b). Notably, PT109B administration in normal mice had no discernible impact on behavior. Motor function improvements were further evidenced by the pole climbing assay, where PT109B-treated PD mice exhibited substantially reduced climbing times compared to untreated PD mice, with selegiline-treated mice showing a lesser improvement (Fig. 5 c). The rotarod test showed that in PD mice treated with PT109B, the motor coordination ability was enhanced and the endurance time increased, but there was no statistically significant difference found (Fig. 5 d). The hindlimb clasping test further corroborated these findings, with PT109B significantly ameliorating limb posture in PD mice, as reflected by reduced flexion-extension scores (Fig. 5 e). Exploration of anxiety-related behaviors via open field tests revealed that PT109B and selegiline treatments increased central area exploration and movement velocity in PD mice, suggesting slight improvements in both motor function and anxiety (Fig. 5 f-i). Collectively, these results demonstrate that PT109B effectively restores motor function and ameliorates the PD-associated behavioral deficits in vivo . PT109B increases the number of DANs in the brain of PD mice and reprogrammed astrocytes into induced neurons with electrophysiological activity To further elucidate the therapeutic potential of PT109B, we investigated its effects on DAN density and neural circuitry restoration in the substantia nigra and striatum of 6-OHDA-induced PD mice. DANs in the damaged striatum and substantia nigra of the model group showed significant reduction compared to controls, accompanied by marked morphological degeneration, including loss of intact neuronal soma (Fig. 6 a, b). In contrast, the PT109B-treated mice exhibited a pronounced increase in DAN density in these regions, with preserved neuronal morphology (Fig. 6 a,b). Immunofluorescence analysis further demonstrated elevated levels of NeuN + and TH + cells in PT109B-treated mice, indicating not only enhanced DAN numbers but also the restoration of neuronal integrity. In comparison, selegiline treatment failed to significantly restore DAN density or improve neuronal morphology (Fig. 6 a-e). To assess whether PT109B could reprogram astrocytes into functional neurons in vivo , we injected AAV5-GFAP-EGFP into the striatum and substantia nigra to label astrocytes, followed by oral administration of PT109B (100 mg/kg) for 3 months. While GFP + cells with neuron-like morphology were observed, no significant co-labeling of GFP + and NeuN + cells was detected (Fig. 6 f), suggesting incomplete reprogramming of astrocytes into mature neurons. To evaluate the restoration of dopaminergic circuitry, we injected the retrograde tracer CTB into the striatum. Control mice exhibited robust CTB and TH co-labeling in the substantia nigra, indicative of intact dopaminergic projections. In contrast, PD model mice showed diminished CTB signals and significant DAN loss. Remarkably, PT109B treatment restored both CTB signals and DAN density, with co-labeling of CTB and TH, suggesting the structural recovery of dopaminergic projections (Fig. 6 g, h). Electrophysiological recording of GFP-labeled cells in the striatum revealed two distinct populations: (i) cells with very small cell bodies lacking APs and sodium-potassium currents, consistent with astrocytes; and (ii) larger and rounded cells exhibiting neuronal activity are capable to generate repetitive APs and show large voltage-gated sodium and potassium currents (Fig. 6 i-l). These findings suggest that PT109B may partially induce neuronal-like properties in astrocytes without achieving full neuronal conversion. To assess the safety of long-term PT109B administration, we conducted chronic toxicity studies in both C57BL/6 and PD mice. Oral administration of PT109B (100 mg/kg) for 3 months did not affect body weight or organ indices (heart, liver, spleen, lungs, kidneys, and brain) in any group (Fig. S6a, b). Histopathological examination (H&E staining) revealed no evidence of organ damage, confirming the absence of pathological changes (Fig. S6c, d). Liver function assays indicated no significant alterations in alanine aminotransferase (ALT), aspartate aminotransferase (AST), or gamma-glutamyl transferase (γ-GT) levels (Fig. S6e-g). Similarly, kidney function markers, including blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA), remained unchanged across all groups (Fig. S6h-j). Hematological analysis revealed no significant differences in white blood cells, neutrophil, lymphocytes, or monocyte counts (Fig. S6k-n). Red blood cell parameters, including mean corpuscular volume, mean corpuscular hemoglobin, and red blood cell count, were unaffected by PT109B (Fig. S6o-q). While hemoglobin levels were slightly elevated in PT109B-treated mice, these changes were not statistically significant compared to controls (Fig. S6r). Platelet count and mean platelet volume also remained unaltered (Fig. S6s, t). Together, these results demonstrate that PT109B exhibits an excellent safety profile, supporting its potential for long-term therapeutic use in PD. Discussion In this study, we identified PT109B as a potent inducer of reprogramming astrocytes into functional iDANs. Remarkably, PT109B initiated astrocytes reprogramming within 3 hours. The reprogrammed iDANs exhibited synaptic structures, neuron-like electrophysiological activity, dopamine release capabilities, and long-term survival (for over 3 months) in vitro . Importantly, oral administration of PT109B in a 6-OHDA-induced PD mouse model not only alleviated motor deficits but also increased the number of dopaminergic neurons in the striatum and substantia nigra. By overcoming key bottlenecks in chemical reprogramming and demonstrating therapeutic potential in PD models, PT109B provides a promising foundation for developing tools useful for regenerative therapies aiming at for neurodegenerative diseases. Compared to mature neurons, astrocytes possess a lower resting membrane potential (approximately − 90 mV) and reduced membrane resistance. The large surface area and low membrane resistance of astrocytes make the detection of AP inherently challenging[ 29 ]. In this study, the single AP, generated by PT109B-induced cells, were of modest amplitude, with relatively small sodium currents, indicating the presence of functional sodium and potassium channels and the initiation of depolarization. While the degree of depolarization was limited, these changes significantly altered the electrophysiological profile of astrocytes, marking a critical step toward neuronal maturation. The Na-K-ATPase, a membrane protein essential for regulating intracellular and extracellular sodium and potassium ion concentrations, plays a pivotal role in maintaining the osmotic balance and facilitating depolarization and hyperpolarization in cells[ 30 ]. We hypothesize that the inability of PT109B to induce continuous AP may stem from insufficient Na-K-ATPase activity in the reprogrammed cells. Supporting this, T3, a biologically active hormone, has been shown to specifically enhance Na-K-ATPase activity. T3 (50 nM) promotes changes in the extracellular matrix of astrocytes[ 31 ]. To address the limitations in PT109B-induced electrophysiological activity, we supplemented the induction system with T3 (50 nM). This combination markedly increased the proportion of cells exhibiting continuous AP after 15 or 30 days of treatment (Fig. 2 m). Additionally, the amplitude of sodium currents significantly increased in bipolar or tripolar induced neurons with smaller cell bodies, and some cells displayed excitatory postsynaptic electrophysiological properties. These results indicate that T3 synergistically enhances the electrophysiological maturation of PT109B-induced neurons, enabling the detection of continuous AP in treated astrocytes. Notably, the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) using chemical small molecules has been shown to involve cell cycle arrest, a state characterized by paused cell division and delayed development. During this process, histone protein acetylation increases, enhancing chromatin accessibility and promoting reprogramming efficiency. Similarly, modulating cell proliferation, such as by knocking out p53 in somatic cells, including fibroblasts and astrocytes, has been demonstrated to significantly enhance the reprogramming efficiency of transcription factors[ 32 ]. PT109B has been shown to induce cell cycle arrest in glioma cells, promoting their transformation into neuron-like cell[ 18 ]. These parallels suggest that the cell cycle arrest state induced by PT109B is a key factor facilitating the direct conversion of astrocytes into neurons. The emergence of intermediate neuron-like cells (MAP2 + /TH + /GFAP + ) during PT109B-induced reprogramming further supports the notion that astrocytes can be directly converted into neurons. Given the shared lineage and functional similarities between astrocytes and neurons, this direct conversion is biologically plausible[ 33 ]. The appearance of intermediate cells expressing both neuronal and glial markers suggests a transitional state during reprogramming, wherein astrocytes progressively adopt neuronal characteristics while shedding their glial identity. Our findings reveal that PT109B induces profound morphological changes, upregulates neuronal markers, and enhances cellular activation and oxidative stress levels during the reprogramming process. These effects are mediated, in part, by the upregulation of b-HLH family transcription factors, including Ascl1, Ngn2, and NeuroD1. These factors are known to play critical roles in neuronal differentiation and maturation. Previous studies have demonstrated that the combinations of chemicals can reprogram astrocytes into neurons within 2–4 days, accompanied by dynamic changes in b-HLH family transcription factors (Ascl1, NeuroD1, and Ngn2)[ 15 ]. Consistent with this, we observed that PT109B significantly elevated the protein levels of these transcription factors within 6 hours, while simultaneously reduced the expressions of inhibitory proteins PTBP1 and REST. This rapid molecular response is accompanied by a marked activation of astrocytes. Although PTBP1 knockdown has been reported to reprogram astrocytes into DANs, recent studies have cast doubt on its role as a gatekeeper gene in this process[ 20 , 34 ]. While we initially hypothesized that PTBP1 might be a key mediator of PT109B's effects, the overexpression of PTBP1 in PT109B-treated astrocytes failed to reverse the morphological changes induced by the compound. However, short-term transcriptomic analysis revealed that the PTBP1 pathway is may not a primary target of PT109B, suggesting that other mechanisms could account its reprogramming efficacy. Our RNA sequencing data highlights the upregulation of pathways associated with cellular activation, including TGF-β, MAPK, and TNF-α, within 6 hours of PT109B treatment (Fig. 4 k, l). Conversely, the cell cycle pathway is significantly suppressed. These findings align with previous reports that overexpression of b-HLH family transcription factors induces a state of high oxidative stress and cell cycle arrest during neuronal reprogramming[ 25 ]. The MAPK cascade, a central hub for signal transduction, regulates diverse cellular processes, including proliferation, differentiation, and apoptosis. In unstimulated cells, MAPK remains inactive; however, stimulation triggers its activation via MKK and MKKK-mediated phosphorylation[ 35 ]. PT109B treatment significantly enhances MAPK pathway activity, indicating a highly activated state in treated astrocytes. This activation coincides with elevated expression of neural stem cell genes, and elevated levels of inflammatory factors like TNF-α and TGF-β1[ 36 ]. These changes suggest that astrocytes acquire a stem-like state, characterized by an open chromatin conformation that facilitates gene expression. These findings suggest that PT109B-induced TNF-α elevation plays a pivotal role in astrocytes activation and dedifferentiation, warranting further investigation into the precise signaling mechanisms involved. Interestingly, PT109B also significantly increases TGF-β1 levels, alongside elevated BMP1 and SMAD3 expression. TGF-β1, primarily sequestered in the extracellular matrix, is released in response to extracellular oxygen radicals or integrin signaling. Upon binding to the TGF-βR1/2 receptor complex, it triggers SMAD2/3 phosphorylation and subsequent nuclear translocation of SMAD2/3-SMAD4 complexes, regulating genes involved in cell activation and differentiation[ 37 ]. Whether the TGF-β pathway is a key mediator of PT109B's effects remains to be explored. Epigenetic reprogramming mechanism is likely enhanced by small chemical molecules, which promote DNA accessibility and transcriptional activation[ 38 ]. Recent advancements in epigenomic profiling, including ATAC-seq, histone modification omics, and DNA methylation analyses, have identified key targets of small molecule-mediated reprogramming[ 39 ]. Applying these techniques in future studies could elucidate the epigenetic changes driving PT109B-induced astrocytes reprogramming and identify its molecular targets. To further evaluate PT109B therapeutic potential for PD in vivo , we employed the 6-OHDA-induced PD mouse model, which exhibits high DAN mortality, and irreversible PD symptoms[ 40 ]. Our results demonstrate that PT109B administration significantly ameliorates PD-associated behavioral deficits, including anxiety-like behavior, reduced motor speed, endurance, and impaired coordination. Furthermore, PT109B increased the number of DANs in the lesioned striatum of PD mice. To investigate the fate of astrocytes in vivo , we utilized AAV5-GFAP-EGFP to specifically label astrocytes in the brains of PD mice. While GFP + cells exhibited morphological and electrophysiological characteristics resembling neurons, we did not observe co-localization of GFP with the mature neuronal marker NeuN (Fig. 5 f). This suggests that PT109B may not induce fully reprogram of astrocytes into mature neurons but rather it may induce an immature neuron-like state. To test this hypothesis, future studies could assess the co-expression of GFP with immature neuronal markers such as DCX. However, limitations associated with AAV-based tracing, including potential leakage and loss of fluorescence labeling and the influence of transcription factor levels on GFAP promoter activity, underscore the need for more reliable lineage-tracing tools[ 41 , 42 ]. Future studies utilizing astrocytes lineage tracing system, such as Aldh1l1-CreERT2 mice may provide more definitive insights into the in vivo reprogramming effects of PT109B. Additionally, we employed retrograde tracing with CTB injected into the striatum to monitor dopaminergic projections. Compared to the control group, PT109B-treated PD mice exhibited significant CTB + /TH + co-staining in the substantia nigra, indicating partial recovery of the nigrostriatal dopaminergic pathway (Fig. 4 g, h). This finding is consistent with the observed behavioral improvements and increased DAN counts, further supporting the neurorestorative potential of PT109B. Conclusions Together, PT109B is shown to be a promising small molecule that can reprogram astrocytes into functional iDANs, alleviate PD-related motor deficits and contribute to the restoration of dopaminergic neuron populations, presenting an alternative option for the treatment of PD. Abbreviations PD Parkinson's disease iDANs Induced dopaminergic neurons b-HLH Basic Helix-Loop-Helix DANs Dopaminergic neurons 6-OHDA 6-hydroxydopamine HP-β-CD Hydroxypropyl-β-cyclodextrin AP Anterior-posterior ML Medio-lateral DV Dorso-ventral CTB Cholera toxin B ACSF Artificial Cerebrospinal Fluid DEGs Differentially expressed genes GO Gene ontology SD Sprague-Dawley PBS Phosphate-buffered saline DAPI 4’,6-diamidino-2-phenylindole TMB 3,3’,5,5’-tetramethylbenzidine PCA Principal component analysis MFB Medial forebrain bundle Declarations Acknowledgements We extend our gratitude to Prof. Zhongwei Zhou for generously providing the mouse primary neural stem cells. Our appreciation also goes to the Guangdong Provincial Key Laboratory of Brain Function and Disease for their invaluable guidance and support in confocal microscopy. We are deeply thankful to Prof. Gao Jin and Dr. Leung Ka Wing from the Division of Life Science and the Center for Chinese Medicine at The Hong Kong University of Science and Technology. Their expertise and assistance were instrumental in the design and execution of the animal experiments. Graphical abstract was created by Biorender.com. Author contributions Y.Y., C.L.W., and Z.Y.Z designed the overarching concepts for the study. T.H.F., J.W.Z., and Y.L.T planned the research methods. S.S.W. developed the necessary software tools. C.L.W., Y.L., D.C., and Z.X.Z. carried out validation processes. Y.Y., C.L.W., and Y.R. conducted formal analyses. C.L.W., Y.Y., K.J., and S.Q.H. were involved in research investigations. W.B.D., Q.Z., and C.L. provided the required resources. Y.L. and Y.P.C. curated the research data. C.L.W., Y.Y., and T.H.F. designed experiments for the initial draft and wrote it. R.B.P. and K.W-K.T. helped refine the manuscript with their review and editing work. Y.Y., C.L.W., S.S.W., and Z.L. designed visualizations and helped present the data. R.B.P. supervised the overall research process. R.B.P. and K.W-K.T. managed the project. R.B.P. acquired the necessary funding for the project. Funding This work was supported by the Shenzhen Science and Techonlogy Program (Grant No. 202111233000079) to R.B.P. Availability of data and materials All RNA-seq raw data and count files were deposited in the Gene Expression Omnibus as a Super Series under accession number GEO: GSE292869. Raw western blot data are deposited in Mendeley Data (DOI:10.17632/gmd3t57fnt.1) Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request. Ethics approval and consent to participate All animal experiments conducted were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University, and were performed under SYSU-IACUC-2021-B0137. Clinical trial number: not applicable. Consent for publication Not applicable. Competing interests All authors declare that there are no conflicts of interest. Author details 1 School of Medicine, Sun Yat-sen University - Shenzhen Campus, Shenzhen 518107, China. 2 School of Pharmacy/Key Laboratory of Xinjiang Phytomedicine Resource and Utilization Ministry of Education/Institute for Safflower Industry Research, Shihezi University, Shihezi 832000, China. 3 School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China. 4 School of Life Sciences, South China Normal University, Guangzhou 510631, China. 5 School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China. 6 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong 999077, China. 7 School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University - Shenzhen Campus, Shenzhen 518107, China. 8 Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong 999077, China. 9 International Joint Laboratory (SYSU-PolyU HK) of Novel Anti-Dementia Drugs of Guangdong, Guangzhou 510006, China. 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Supplementary Files TableS1siRNAsusedinthisstudy.xlsx TableS2Primersusedinthisstudy.xlsx Graphicabstract.docx Supplementaryfigure.docx originaldataofWB.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6428230","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446614960,"identity":"c8bd35f8-30f7-44ea-9a68-abf2038f3d7d","order_by":0,"name":"Cailv Wei","email":"","orcid":"","institution":"Sun Yat-sen University - Shenzhen Campus","correspondingAuthor":false,"prefix":"","firstName":"Cailv","middleName":"","lastName":"Wei","suffix":""},{"id":446614961,"identity":"078ea346-2f6f-4991-813a-bf3e2986e368","order_by":1,"name":"Yang Yang","email":"","orcid":"","institution":"Shihezi University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Yang","suffix":""},{"id":446614962,"identity":"4347a755-7090-43b6-94ee-55d615eed6ed","order_by":2,"name":"Tsz Hei Fong","email":"","orcid":"","institution":"Peking University Shenzhen Graduate School","correspondingAuthor":false,"prefix":"","firstName":"Tsz","middleName":"Hei","lastName":"Fong","suffix":""},{"id":446614963,"identity":"8bbcd161-d155-45eb-afe0-23a03431daf0","order_by":3,"name":"Yuan Liu","email":"","orcid":"","institution":"Sun Yat-sen University - 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Shenzhen Campus","correspondingAuthor":true,"prefix":"","firstName":"Rongbiao","middleName":"","lastName":"Pi","suffix":""}],"badges":[],"createdAt":"2025-04-11 12:11:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6428230/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6428230/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81370101,"identity":"0b82bdcd-1ef4-441b-b043-c83cf47a972b","added_by":"auto","created_at":"2025-04-25 10:20:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2921695,"visible":true,"origin":"","legend":"\u003cp\u003ePT109B rapidly reprogrammed astrocytes into induced neurons. \u003cstrong\u003ea \u003c/strong\u003eThe primary rat midbrain astrocytes were exposed to PT109B (10 μM) for different time: Test 1 (1, 3, and 7 days); Test 2 (0.75, 1.5, 3, 6, 12, 24, and 72 hours). \u003cstrong\u003eb, c\u003c/strong\u003e Morphological changes of astrocytes after PT109B treatment for 1, 3, 7 days. \u003cstrong\u003eb\u003c/strong\u003e Representative images. Scale bar = 20 μm. \u003cstrong\u003ec\u003c/strong\u003e Key morphological parameters: branch length, branch number, and cell territory area. \u003cstrong\u003ed-f \u003c/strong\u003eStatistical analysis of (\u003cstrong\u003ed\u003c/strong\u003e) branch length, (\u003cstrong\u003ee\u003c/strong\u003e) branch number, and (\u003cstrong\u003ef\u003c/strong\u003e) cell territory area. (n=10). \u003cstrong\u003eg\u003c/strong\u003e Western blot analysis the protein level of astrocytes marker (GFAP), mature neuron markers (MAP2, NeuN) and dopaminergic neuron markers (DAT and TH) after PT109B treatment for 1, 3, 7 days. \u003cstrong\u003eh\u003c/strong\u003e Quantification of GFAP, MAP2, NeuN, DAT, and TH protein levels. (n=3). \u003cstrong\u003ei\u003c/strong\u003e Western blot analysis the protein level of activated astrocytes marker (C3, TGF-β1), oxidative stress (COX2) and inflammatory factors (TNF-α). \u003cstrong\u003ej\u003c/strong\u003e Quantification of C3, TGF-β1, COX2 and TNF-α protein levels. (n=3). \u003cstrong\u003ek\u003c/strong\u003e Morphology changes of astrocytes after PT109B treatment for 0, 0.75, 1.5, 3, 6, 12, 24, and 72 hours. Scale bar = 10 μm. \u003cstrong\u003el\u003c/strong\u003e Western blot analysis the protein level of immature neuron maker DCX and Tuj1. \u003cstrong\u003em\u003c/strong\u003e Quantification of DCX and Tuj1 protein level. (n=3). \u003cstrong\u003en\u003c/strong\u003e Immunofluorescence showing GFAP\u003csup\u003e+\u003c/sup\u003e and Tuj1\u003csup\u003e+\u003c/sup\u003e astrocytes after PT109B treatment for 3 days. Scale bar = 20 μm. \u003cstrong\u003eo\u003c/strong\u003e Quantification of Tuj1\u003csup\u003e+\u003c/sup\u003e cells percentage. (n=3). Data are reported as mean ± SEM; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/4b612a86721022aa9b58ac15.png"},{"id":81369605,"identity":"2eb4f8b1-5a5b-49bc-bcc6-2c0c9f84de8e","added_by":"auto","created_at":"2025-04-25 10:12:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3357950,"visible":true,"origin":"","legend":"\u003cp\u003ePT109B reprogrammed astrocytes into functional iDANs.\u003cstrong\u003e a, b\u003c/strong\u003e Left: immunofluorescence staining of NeuN and co-stained with (\u003cstrong\u003ea\u003c/strong\u003e) MAP2, (\u003cstrong\u003eb\u003c/strong\u003e) TH in astrocytes treated with PT109B for half a month. Scale bar = 20 μm; right: quantification of (\u003cstrong\u003ea\u003c/strong\u003e) MAP2\u003csup\u003e+\u003c/sup\u003e and NeuN\u003csup\u003e+\u003c/sup\u003e cells or (\u003cstrong\u003eb\u003c/strong\u003e) TH\u003csup\u003e+\u003c/sup\u003e cells percentages. (n=3). \u003cstrong\u003ec-f\u003c/strong\u003e Left: immunofluorescence analysis of (\u003cstrong\u003ec\u003c/strong\u003e) DAT, (\u003cstrong\u003ed\u003c/strong\u003e) Nurr1, (\u003cstrong\u003ee\u003c/strong\u003e) Synap1 and (\u003cstrong\u003ef\u003c/strong\u003e) NaV in astrocytes treated with PT109B for 1 month. Scale bar = 20 μm; right: quantification of cell percentages. (n=3). \u003cstrong\u003eg\u003c/strong\u003e Morphological changes and immunofluorescence staining of MAP2, NeuN and TH in cultured astrocytes treated with PT109B for 0, 1, 2, and 3 months. Scale bar = 10 μm. \u003cstrong\u003eh\u003c/strong\u003e Left: representative traces of membrane voltages elicited by depolarizing current steps, recorded in cultured cells treated with PT109B for 0, 1, 2, and 3 months; right: changes in inward/outward currents with increasing voltage steps (-70 mV to 70 mV, step by 10 mV, 500 ms duration, holding at -70 mV). \u003cstrong\u003ei\u003c/strong\u003e Measurement of dopamine content in astrocytes exposed to PT109B for 1 month. (CT, n=5; PT109B, n=4) \u003cstrong\u003ej\u003c/strong\u003e Quantification of peak amplitudes in PT109B-treated cells at different time point. (CT, n=11; PT109B 1Mon, n=15; 2Mon, n=11; 3Mon, n=6). \u003cstrong\u003ek\u003c/strong\u003e Cell capacitance before and after PT109B treatment. (CT, n=11; PT109B n=28). \u003cstrong\u003el\u003c/strong\u003e Percentage of PT109B-treated cells showing depolarized peak in recorded neuron-like cells. (PT109B 1Mon, n=40; 2Mon, n=36; 3Mon, n=16). \u003cstrong\u003em\u003c/strong\u003e Percentage of iDANs exhibiting repetitive (multiple), single, or no AP with 0.5- or 1-month PT109B+T3 treatment. (PT109B+T3, 0.5 Mon, n=63; 1Mon, n=42). \u003cstrong\u003en\u003c/strong\u003e Input resistance of recorded cells without treatment, with PT109B only, or with PT109B+T3. (CT, n=24; PT109B, n=22; PT109B+T3, n=51). \u003cstrong\u003eo, p\u003c/strong\u003e Representative images of recorded iDANs with PT109B+T3 treatment for (\u003cstrong\u003eo\u003c/strong\u003e) 0.5 or (\u003cstrong\u003ep\u003c/strong\u003e) 1 month. \u003cstrong\u003eq, r\u003c/strong\u003e iDANs showing repetitive AP (left), voltage-dependent inward currents (middle), and spontaneous postsynaptic currents (right) with PT109B+T3 treatment for (\u003cstrong\u003eq\u003c/strong\u003e) 0.5 or (\u003cstrong\u003er\u003c/strong\u003e) 1 month. Data are reported as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/fd90bbcd60bc8d92495036e1.png"},{"id":81369608,"identity":"3893b69b-a9a9-41fc-8d1a-b45e10c588c1","added_by":"auto","created_at":"2025-04-25 10:12:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":959366,"visible":true,"origin":"","legend":"\u003cp\u003ePT109B enhances b-HLH family transcription factors to drive neuronal reprogramming.\u003cstrong\u003e \u003c/strong\u003ePrimary rat midbrain astrocytes were treated with PT109B (10 μM) for different time (0, 3, 6, 12, 24, 72, and 168 hours). \u003cstrong\u003ea, b\u003c/strong\u003e QPCR analysis of (\u003cstrong\u003ea\u003c/strong\u003e) the neuronal markers MAP2, NeuN, Tuj1, and DCX, (\u003cstrong\u003eb\u003c/strong\u003e) and key transcription factors, including NeuroD1, Ngn2, Nurr1, Ascl1, and Dlx2 mRNA expression levels after 72 hours of PT109B treatment. (n=3). \u003cstrong\u003ec\u003c/strong\u003e QPCR analysis of MAP2, TH and NeuroD1 mRNA expression levels after 168 hours of PT109B treatment. (n=3). \u003cstrong\u003ed\u003c/strong\u003e Western blot analysis of the MAP2, Dlx2, Ngn2, Ascl1, Nurr1, and NeuroD1. \u003cstrong\u003ee\u003c/strong\u003eQuantification of MAP2 and b-HLH family transcription factors protein levels. (n=3). \u003cstrong\u003ef\u003c/strong\u003e Western blot analysis of REST/PTBP1 pathway-related proteins (REST, PTBP1) and other transcription factors (Brn2, c-Myc, Lhx8, Isl-1, OCT4) involved in neuronal reprogramming. \u003cstrong\u003eg\u003c/strong\u003e Quantification of REST, PTBP1, c-Myc, Isl-1, Brn2, and Lhx8 protein levels. (n=3). Data are reported as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/c5b1b29ab71923a63f1983fa.png"},{"id":81370928,"identity":"bbb97e9c-e05a-46a7-8ee3-e0b5b1e407ee","added_by":"auto","created_at":"2025-04-25 10:28:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1534940,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptomic profiling reveals early mechanisms of PT109B-induced astrocytes reprogramming\u003cstrong\u003e. a\u003c/strong\u003e PCA of astrocytes at distinct stages of PT109B treatment. (CT, n=3; 1.5 h, n=2; 3 h, n=2; 6 h, n=2). \u003cstrong\u003eb\u003c/strong\u003e PCA of all samples, with a solid line tracing the trajectory of transcriptional changes during PT109B-induced reprogramming. \u003cstrong\u003ec\u003c/strong\u003e Bar plot of up- or down-regulated DEGs in PT109B-treated astrocytes vs. controls (adjusted \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, fold change \u0026gt; 2). \u003cstrong\u003ed\u003c/strong\u003e GO analysis of DEGs at different time points, highlighting enriched biological processes. \u003cstrong\u003ee\u003c/strong\u003e Top ten significantly enriched KEGG pathways at 6 hours PT109B treatment. \u003cstrong\u003ef-h\u003c/strong\u003e Volcano plots of significantly altered genes at (\u003cstrong\u003ef\u003c/strong\u003e) 1.5 hours, (\u003cstrong\u003eg\u003c/strong\u003e) 3 hours, and (\u003cstrong\u003eh\u003c/strong\u003e) 6 hours (red: up-regulated, blue: down-regulated; adjusted \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, fold change \u0026gt; 2). \u003cstrong\u003ei\u003c/strong\u003eGSEA of 6 hours PT109B-treated samples vs. controls. \u003cstrong\u003ej\u003c/strong\u003e Heatmap of leading-edge gene subsets corresponding to enriched pathways in (\u003cstrong\u003ei\u003c/strong\u003e) (red: high expression, blue: low expression). \u003cstrong\u003ek\u003c/strong\u003e Identification of three major gene clusters (green, red, blue) representing distinct functional modules in reprogramming.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/cd176b5b1e4c8e9e13154c1b.png"},{"id":81369610,"identity":"43fc1473-d6e6-4876-bbac-98c3f7ea2789","added_by":"auto","created_at":"2025-04-25 10:12:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1149444,"visible":true,"origin":"","legend":"\u003cp\u003ePT109B ameliorates motor dysfunction in 6-OHDA induced PD mice. \u003cstrong\u003ea\u003c/strong\u003e Schematic timeline of the experimental design. Mice underwent unilateral stereotactic injection of 6-OHDA into the MFB to induce PD. After 3 weeks, AAV5-GFAP-EGFP was injected into the striatum and substantia nigra to label astrocytes. Mice were orally administered PT109B (100 mg/kg) or selegiline (10 mg/kg) for 12 weeks. Behavioral tests and immunofluorescence were performed at 6 weeks, and CTB retrograde tracer was injected 1 week before sacrifice. \u003cstrong\u003eb\u003c/strong\u003e Apomorphine-induced rotation test (0.5 mg/kg, i.p.). \u003cstrong\u003ec\u003c/strong\u003e Pole test. \u003cstrong\u003ed\u003c/strong\u003e Rotarod test. \u003cstrong\u003ee\u003c/strong\u003e Hindlimb clasping test. \u003cstrong\u003ef-i\u003c/strong\u003e Open field test. \u003cstrong\u003ef\u003c/strong\u003e Heatmap of movement patterns. \u003cstrong\u003eg\u003c/strong\u003e Time spent in the center zone. \u003cstrong\u003eh\u003c/strong\u003e Movement speed. \u003cstrong\u003ei\u003c/strong\u003e Total distance traveled. Control group (CT), n = 12, Model, n = 10, Model+PT109B, n = 12, Model+Selegiline, n = 10, Sham, n = 8, Sham+PT109B, n = 9, Sham+Selegiline, n = 13. Data are reported as mean ± SEM, ns = no significance, *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/6539aebdacae1234132f0e7c.png"},{"id":81369613,"identity":"6669c601-0e53-406a-885f-5c6b6af11427","added_by":"auto","created_at":"2025-04-25 10:12:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6082865,"visible":true,"origin":"","legend":"\u003cp\u003ePT109B restores dopaminergic neurons and neural circuitry in 6-OHDA-induced PD mice. \u003cstrong\u003ea, b\u003c/strong\u003e Immunofluorescence of TH\u003csup\u003e+\u003c/sup\u003e dopaminergic neurons in the (\u003cstrong\u003ea\u003c/strong\u003e) striatum and (\u003cstrong\u003eb\u003c/strong\u003e) substantia nigra of mice after 12 weeks of PT109B (100 mg/kg) or selegiline (10 mg/kg) treatment. Scale bars: large images = 500 µm, insets = 50 µm. \u003cstrong\u003ec\u003c/strong\u003e Co-immunostaining of NeuN\u003csup\u003e+\u003c/sup\u003e neurons (green) and TH\u003csup\u003e+\u003c/sup\u003e dopaminergic neurons (red) in the striatum and substantia nigra. Scale bars = 50 µm. \u003cstrong\u003ed-e\u003c/strong\u003e Quantitative analysis of (\u003cstrong\u003ed\u003c/strong\u003e) TH\u003csup\u003e+\u003c/sup\u003e and (\u003cstrong\u003ee\u003c/strong\u003e) NeuN\u003csup\u003e+\u003c/sup\u003e cell ratios. (n=4). \u003cstrong\u003ef\u003c/strong\u003e Three-dimensional reconstruction of GFP\u003csup\u003e+\u003c/sup\u003e cells showing neuron-like morphology. \u003cstrong\u003eg\u003c/strong\u003e Experimental timeline. Mice received unilateral stereotactic injection of 6-OHDA into the MFB and were monitored for 3 weeks. PT109B (100 mg/kg) was administered orally for 12 weeks, starting at day 28. At day 110, the retrograde tracer CTB was injected into the striatum. \u003cstrong\u003eh\u003c/strong\u003e Immunostaining of TH (red), CTB (green), and DAPI (blue) in the substantia nigra, with TH-CTB co-localization indicating restored dopaminergic projections. \u003cstrong\u003ei\u003c/strong\u003e Representative GFP\u003csup\u003e+\u003c/sup\u003e cell (green) for patch-clamp recording. Scale bar = 10 µm. (n=22). (J-K) Electrophysiological recordings of GFP-labeled cells in the striatum of PT109B-treated mice, revealing \u003cstrong\u003ej\u003c/strong\u003e AP and \u003cstrong\u003ek\u003c/strong\u003e sodium-potassium currents. \u003cstrong\u003el\u003c/strong\u003e Different populations of recorded cells in striatum. Data are reported as mean ± SEM. *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 vs. control group.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/6942c4138c79f8aacf9bb4d6.png"},{"id":83876582,"identity":"fcdc6dc7-c05e-4303-ae21-c8c850855d90","added_by":"auto","created_at":"2025-06-04 03:43:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19137776,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/c5e2215e-c245-4a4b-bd36-b07b9a1c3df7.pdf"},{"id":81369603,"identity":"de81cee3-d5fc-46a7-9a41-c9551f902fc0","added_by":"auto","created_at":"2025-04-25 10:12:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9628,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1siRNAsusedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/3a76e9b58a994dd3327af2e4.xlsx"},{"id":81369604,"identity":"fd0a01b4-07c6-440e-8219-bac59a8177a4","added_by":"auto","created_at":"2025-04-25 10:12:02","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11421,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2Primersusedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/55ac2772620474cb5df0cd09.xlsx"},{"id":81369625,"identity":"00dcf687-e9ff-4d24-a6e0-963c2b48f8a2","added_by":"auto","created_at":"2025-04-25 10:12:03","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":525911,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/80727f3e3290e3749df29271.docx"},{"id":81370105,"identity":"6fdf7179-3423-4dc4-99cb-6b419f662316","added_by":"auto","created_at":"2025-04-25 10:20:03","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9423691,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/1f24eac46a8a512995d2f656.docx"},{"id":81370103,"identity":"0fa98a29-0e4d-43db-8403-f94c4773b51d","added_by":"auto","created_at":"2025-04-25 10:20:02","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2183671,"visible":true,"origin":"","legend":"","description":"","filename":"originaldataofWB.docx","url":"https://assets-eu.researchsquare.com/files/rs-6428230/v1/9837740ac7f4b2267795de10.docx"}],"financialInterests":"","formattedTitle":"PT109B, a Multikinase Inhibitor, Converts Astrocytes into Dopaminergic Neurons and Alleviates Parkinson's Disease in Mice","fulltext":[{"header":"Background","content":"\u003cp\u003eParkinson's disease (PD) is a progressive neurodegenerative disease characterized by motor dysfunction, with the pathological hallmark being the selective loss of dopaminergic neurons (DANs) in the substantia nigra pars compacta[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Current pharmacological treatments, including levodopa and selegiline, primarily alleviate symptoms but fail to halt disease progression or prevent neuronal loss[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This limitation underscores the urgent need for innovative therapeutic strategies capable of regenerating DANs to address the underlying pathology of PD.\u003c/p\u003e \u003cp\u003eAstrocytes, the most abundant non-neuronal cell population in the brain, share a common neuroectodermal origin with neurons and exhibit shared region-specific transcriptional and epigenetic characteristics[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent advances in regenerative medicine have highlighted the potential of reprogramming resident astrocytes into neurons as a promising approach for PD treatment[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Gene therapy-based reprogramming, having effective in preclinical models, faces significant translational challenges, including the risk of gene mutations, genetic toxicity, and off-target effect[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, chemical reprogramming offers a non-genotoxic alternative with substantial clinical potential[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Although small molecule combinations have demonstrated the ability to transform astrocytes into neurons in the models of neurodegenerative diseases[\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], this approach is hindered by several limitations, including the complexity of multi-component systems, low reprogramming efficiency, and poorly understood mechanisms[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, identifying a single small molecule capable of efficiently reprogramming astrocytes into functional neurons will not only deepen our understanding of cell fate plasticity but also provide a promising approach for PD therapeutics.\u003c/p\u003e \u003cp\u003eWe unexpectedly discovered that PT109B, a multi-kinase inhibitor, promotes neurogenesis in C17.2 cells and enhances synaptogenesis in primary cultured rat hippocampal neurons, and it also induces glioblastoma differentiation via the PTBP1/PKM1/PKM2 pathway[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition to its biological activity, PT109B demonstrates favorable pharmacokinetic properties, including a half-life of 3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 hours and efficient blood-brain barrier penetration[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], positioning it as a promising therapeutic candidate for central neurodegenerative diseases. In this study, we developed a streamlined protocol using PT109B as a single-agent inducer to rapidly and directly reprogram astrocytes into functional induced dopaminergic neurons (iDANs). Remarkably, \u003cem\u003ein vivo\u003c/em\u003e application of PT109B not only alleviated motor deficits in a 6-hydroxydopamine (6-OHDA)-induced PD mouse model, but also increased the number of DANs in the striatum and substantia nigra. Our findings identify that PT109B as a novel small molecule with the capacity to efficiently reprogram astrocytes into iDANs, presenting a substitute approach for the treatment of neurodegenerative disorders.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal\u003c/h2\u003e \u003cp\u003e8-week-old male C57BL/6 mice were obtained from Zhuhai BesTest Bio-Tech Co. and used to evaluate the anti-PD effects of PT109B. All animals were housed in a specific pathogen-free facility maintained at a controlled temperature of 21\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ and relative humidity of 65%, under a 12 hours light/dark cycle. Mice were acclimatized for at least 1 week prior to experiments. All experimental procedures were conducted in accordance with the ethical guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of medication\u003c/h3\u003e\n\u003cp\u003ePT109B was prepared as an oral suspension for administration via gavage. Briefly, PT109B was dissolved in physiological saline containing 20% (w/v) hydroxypropyl-β-cyclodextrin (HP-β-CD) to achieve a final concentration of 20 mg/mL. The suspension was freshly prepared and vortexed thoroughly to ensure homogeneity. PD mice models were induced by 6-OHDA prior to treatment. For dosing, mice received 100 mg/kg of PT109B or 10 mg/kg Selegiline administered orally via gavage once daily for a total duration of 3 months.\u003c/p\u003e\n\u003ch3\u003eEstablishment of the 6-OHDA-induced PD mice model\u003c/h3\u003e\n\u003cp\u003eThe 6-OHDA-induced PD mice model was established according to referenced protocols[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Briefly, mice were anesthetized using an isoflurane anesthesia system and securely positioned on a stereotaxic apparatus to stabilize the head. After shaving the surgical site and disinfecting with alcohol or iodine, a midline incision was made on the scalp, and the periosteum was gently removed to expose the skull and achieve hemostasis. The surgical area was further cleaned with 3% hydrogen peroxide, and the bregma point was identified as the anatomical reference. Using the stereotaxic atlas for mice, the coordinates for the mFB were determined as follows: anterior-posterior (AP): -1.2 mm, medio-lateral (ML): +1.3 mm, and dorso-ventral (DV): -4.8 mm. A small burr hole was drilled at the target site, and 6-OHDA solution (4 \u0026micro;g in 2 \u0026micro;L) was injected into the right mFB area using a 10 \u0026micro;L microsyringe at a rate of 0.5 \u0026micro;L/min under dim lighting conditions. The needle was retained for 5 minutes post-injection to minimize backflow and withdrawn slowly over 3 minutes. Successful lesioning was validated by observing contralateral circling behavior, a hallmark of unilateral dopaminergic depletion. Three weeks post-lesioning, mice were screened for successful modeling using a unilateral rotation test induced by subcutaneous administration of apomorphine (0.5 mg/kg). Mice exhibiting\u0026thinsp;\u0026gt;\u0026thinsp;150 contralateral rotations within 30 minutes were considered to have successfully modeled PD and were included in subsequent experiments.\u003c/p\u003e\n\u003ch3\u003eAAV5-GFAP-EGFP labeling of astrocytes in the mouse brain\u003c/h3\u003e\n\u003cp\u003eAstrocytes were labeled in the mouse brain using AAV5-GFAP-EGFP (Shanghai Genechem Co.,Ltd). Mice were anesthetized and securely positioned in a stereotaxic frame. The microinjector was aligned to target the striatum (coordinates: AP: +1.0 mm, ML: +1.6 mm, DV: -2.8 mm) and substantia nigra (coordinates: AP: -3.0 mm, ML: +1.3 mm, DV: -4.35 mm). After stabilizing the injector for 5 minutes to ensure precision, AAV5-GFAP-EGFP (1\u0026times;10\u003csup\u003e12\u003c/sup\u003e vg/mL) was injected into the cortex at a constant rate of 0.2 \u0026micro;L/min for 5 minutes. To minimize reflux, the injector was retained in place for an additional 5 minutes before being slowly withdrawn. The viral construct was sourced from Jikai Gene.\u003c/p\u003e\n\u003ch3\u003eRetrograde labeling of dopaminergic projections using Cholera toxin B (CTB)\u003c/h3\u003e\n\u003cp\u003eRetrograde tracing of dopaminergic neuronal projections from the striatum to the substantia nigra was performed using CTB, conjugated to Alexa Fluor\u0026trade; 488, as previously described[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Mice were anesthetized and mounted on a stereotaxic frame, with the microinjector aligned to the striatum (coordinates: AP: +1.0 mm, ML: +1.6 mm, DV: -2.8 mm). After stabilizing the injector for 5 minutes, CTB (10 mg/mL) was delivered at a constant rate of 0.2 \u0026micro;L/min for 5 minutes. To prevent tracer reflux, the injector was held in place for an additional 5 minutes before being withdrawn slowly.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eApomorphine-induced rotation test\u003c/h2\u003e \u003cp\u003eApomorphine-induced rotation was performed 3 weeks after 6-OHDA lesion and 15 weeks after PT109B or selegiline treatment. Briefly, 10 minutes after intraperitoneal injection of apomorphine (5 mg/kg dissolved in ice-cold saline solution), each mouse was placed in a circular basin, and contralateral rotations were recorded over a 30-minute period.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOpen field test\u003c/h3\u003e\n\u003cp\u003eThe open field test was conducted to evaluate spontaneous locomotor activity, exploratory behavior, and anxiety levels in a novel environment. The apparatus consisted of a square arena measuring 40 \u0026times; 40 \u0026times; 40 cm for mice. Prior to testing, animals were acclimated to the experimental room for 2 hours under consistent lighting and noise conditions to minimize stress. Each animal was gently placed in the center of the arena and allowed to explore freely for 5 minutes. Locomotor parameters, including total distance traveled, time spent in the center zone, distance traveled in the center, and movement speed, were automatically recorded using tracking software. To reduce inter-animal interference, tested individuals were temporarily housed in separate cages. After each session, the arena was thoroughly cleaned with 75% ethanol to eliminate odor cues and allowed to dry before subsequent trials.\u003c/p\u003e\n\u003ch3\u003ePole test\u003c/h3\u003e\n\u003cp\u003eThe pole test was performed as previously described to assess motor coordination and agility in mice[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The apparatus consisted of an 80 cm tall vertical pole (3 cm in diameter) with a circular platform (12 cm diameter) mounted at the top. The platform surface was covered with hemp cloth to provide traction. Each mouse was placed head-down on the platform, and the time taken to descend to the base of the pole was recorded. The test was repeated three times per mouse, and the average descent time was calculated for analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHindlimb clasping test\u003c/h2\u003e \u003cp\u003eMotor impairment was assessed using the hindlimb clasping Test. Mice were gently suspended by the tail, and hind limb movements were scored over a 15-second interval as follows: 0\u0026thinsp;=\u0026thinsp;both hind limbs fully extended (abducted); 1\u0026thinsp;=\u0026thinsp;partial extension of both hind limbs; 2\u0026thinsp;=\u0026thinsp;partial flexion (adduction) of both hind limbs; 3\u0026thinsp;=\u0026thinsp;both hind limbs fully flexed. All tests were video-recorded to ensure accurate scoring and subsequent review.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRotarod test\u003c/h2\u003e \u003cp\u003eMotor coordination and endurance were evaluated using the rotarod test. Mice underwent a 1-day acclimatization training session on the Rotarod apparatus at constant speeds of 10, 15, and 20 rpm for 10 minutes each. During testing, the Rotarod speed was set to an optimal baseline (e.g., 15 rpm), and the latency to fall was recorded over three trials. The average latency across trials was used for statistical analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWhole-cell patch clamp for electrophysiological activity\u003c/h2\u003e \u003cp\u003eWhole-cell patch-clamp recordings were performed on cultured astrocytes/iDANs to assess electrophysiological activity. Artificial Cerebrospinal Fluid (ACSF) was used for cell incubation contained (in mM) 122 NaCl, 2.5 KCl, 1.2 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 24 NaHCO\u003csub\u003e3\u003c/sub\u003e, 12.5 D-glucose, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, 1 MgSO\u003csub\u003e4\u003c/sub\u003e, 1 MgCl\u003csub\u003e2\u003c/sub\u003e, and 5 HEPES. Pipette internal solution (in mM): 128 K-gluconate,10 NaCl, 2 MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 EGTA, 10 HEPES, 0.4 Na\u003csub\u003e2\u003c/sub\u003eGTP, and 4 Na\u003csub\u003e2\u003c/sub\u003eATP. PT109B-induced astrocytes cultured on coverslips were incubated in oxygenated ACSF (95% O₂ and 5% CO₂) for 30 minutes prior to recording. Cells were then transferred to a recording chamber continuously perfused with oxygenated ACSF at room temperature (25℃-30℃).\u003c/p\u003e \u003cp\u003eElectrophysiological Recordings from Brain Slices: Electrophysiological recordings were performed on brain slices to assess neuronal activity. Experimental mice were anesthetized with 1% sodium pentobarbital (intraperitoneal injection) and decapitated. The brain was rapidly removed and immersed in ice-cold, oxygenated slicing solution (95% O\u003csub\u003e2\u003c/sub\u003e and 5% CO\u003csub\u003e2\u003c/sub\u003e) at 0℃. After 1 minute of cooling, the brain was trimmed and mounted on a vibratome stage (Leica VT1200). Brain slices containing the target region were cut at a thickness of 300 \u0026micro;m at a speed of 0.09 mm/s. Slices were transferred to a chamber containing oxygenated ACSF and incubated at 35\u0026deg;C for 30 minutes, followed by an additional 60 minutes at room temperature. The slicing solution was prepared as follows (in mM): 110 choline chloride, 7 MgSO\u003csub\u003e4\u003c/sub\u003e, 2.5 KCl, 1.25 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 25 NaHCO\u003csub\u003e3\u003c/sub\u003e, 25 D-glucose, 11.6 sodium L-ascorbate, 3.1 sodium pyruvate, and 0.5 CaCl\u003csub\u003e2\u003c/sub\u003e. The ACSF for slice incubation contained (in mM) 127 NaCl, 2.5 KCl, 1.25 NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 25 NaHCO\u003csub\u003e3\u003c/sub\u003e, 25 D-glucose, 2 CaCl\u003csub\u003e2\u003c/sub\u003e, and 1 MgCl\u003csub\u003e2\u003c/sub\u003e. The internal potassium-based pipette solution used is the same with cultured cells recordings.\u003c/p\u003e \u003cp\u003eWhole-cell patch-clamp recordings were performed using glass pipettes (5\u0026ndash;8 MΩ) filled with the potasium-based internal solution, and cells were recorded utilizing a HEKA EPC10 dual patch-clamp amplifier (HEKA Elektronik, Germany). Signals were acquired at a sampling rate of 10 kHz and filtered at 2 kHz. For voltage-clamp experiments, cells were held at a holding potential of -70 mV. Recorded data were analyzed offline using Clampfit 11 software (Molecular Devices, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSingle-molecule induced reprogramming of astrocytes into neurons\u003c/h2\u003e \u003cp\u003eThe neuronal induction medius was prepared by combining 50 mL of Neurobasal medium with key supplements: 1% N2 (Gibco, 17502001), 1% B27 (Gibco, A3582801), 1% Glutamax (Gibco, 35050061), and 1% penicillin/streptomycin. To this base medium, the small molecule PT109B (10 \u0026micro;M) and neurotrophic factors\u0026mdash;BDNF (PeproTech, 450-02-50UG), GDNF (PeproTech, 450-10-10UG), b-FGF (PeproTech, 100-18B-50UG)\u0026mdash;were added to generate the PT109B-containing neuronal induction medium. For reprogramming, mature astrocytes were initially cultured for 1\u0026ndash;3 days in a 1:1 mixture of the PT109B-containing neuronal induction medium and high-glucose DMEM. After this transitional phase, the medium was fully replaced with the PT109B-containing neuronal induction medium. astrocytes were maintained in this medium for 14\u0026ndash;90 days, with daily monitoring of cell morphology and viability. The medium was refreshed every 2\u0026ndash;3 days, with the frequency adjusted based on cellular health and metabolic activity. When whole-cell patch clamp was performed to detect electrophysiological function, T3 (Bioss, bs-0339P, 50 \u0026micro;M) was added to neuron induction medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA extracted using TRIzol was submitted to Genedenovo Biotechnology Co., Ltd. (Guangzhou, China) for RNA sequencing. RNA integrity was verified using an Agilent Bioanalyzer, and libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (Illumina). Sequencing was performed on an Illumina NovaSeq 6000 platform, generating paired-end 150 bp reads.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOmics data analysis\u003c/h2\u003e \u003cp\u003eRaw RNA-seq data were processed and analyzed on the Genedenovo multi-omics data mining system platform. Following quality control and read alignment to the reference genome, differentially expressed genes (DEGs) were identified using DESeq2. Gene ontology (GO) enrichment analysis was performed using the DAVID platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://david.ncifcrf.gov/\u003c/span\u003e\u003cspan address=\"https://david.ncifcrf.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Protein-protein interaction networks were constructed using the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and visualized using Cytoscape software (version 3.8.2). Core network modules were identified and analyzed using the MCODE plugin in Cytoscape.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCulture of primary midbrain astrocytes from newborn SD rats\u003c/strong\u003e \u003cp\u003eastrocytes were isolated from the midbrains of 1\u0026ndash;3 days old Sprague-Dawley (SD) rats. Brains were harvested and placed in precooled D-HANKS buffer. Under a microscope, the meninges and blood vessels were meticulously removed to isolate the midbrain, which was then transferred to pre-cooled serum-free DMEM medium. Tissue digestion was performed using 0.125% trypsin for 15 minutes at 37℃, and the reaction was quenched by adding an equal volume of serum-containing medium. After centrifugation at 1,000 rpm for 8 minutes, the supernatant was discarded. The cell pellet was resuspended in serum-containing medium and seeded at a density of 2.0\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/cm\u0026sup2; in astrocytes medium (89% DMEM, 10% FBS, 1% penicillin/streptomycin). The medium was refreshed every three days. On day 7, cultures were subjected to orbital shaking at 37\u0026deg;C and 200 rpm for 12 hours to remove contaminating microglia and oligodendrocytes. The purified astrocytes were then separated using 0.25% trypsin-EDTA and seeded onto poly-L-lysine-coated (0.01 mg/mL) culture plates for subsequent experiments.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCulture of primary cortical neurons from newborn SD rats\u003c/strong\u003e \u003cp\u003ePrimary cortical neurons were isolated from 0\u0026ndash;3 days old SD rat pups. The cortical tissues were excised and minced into approximately 1 mm\u0026sup3; pieces using sterile ophthalmic scissors. The minced tissue was enzymatically digested in 0.125% trypsin and DNase (8 mg/mL) at 37\u0026deg;C for 15 minutes with gentle agitation every 5 minutes. Digestion was halted by adding complete medium. The mixture was centrifuged at 1,000 rpm for 5 minutes, and the supernatant was discarded. The pellet was resuspended in DMEM supplemented with DNase, gently pipetted, and briefly centrifuged at 1,500 rpm to remove cell clumps and debris. The resulting supernatant was centrifuged again at 1,000 rpm for 5 minutes, and the pellet was resuspended in 10% FBS-DMEM. Neurons were counted and seeded at 2\u0026ndash;5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/cm\u0026sup2; onto poly-L-lysine-coated (0.01 mg/mL) dishes or plates. After 2\u0026ndash;4 hours of incubation for cell adherence, the medium was replaced with neurobasal medium supplemented with 1% B27, 0.25 mM glutamine, and 100 U/mL penicillin/streptomycin. Primary neurons were identified on day 3, and experiments commenced on day 7.\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003e \u003cb\u003eCell immunofluorescence staining\u003c/b\u003e: Cells were washed three times with phosphate-buffered saline (PBS) to remove residual serum and cellular debris. After washing, cells were fixed with 4% (w/v) paraformaldehyde in PBS for 30 minutes at room temperature, and permeabilized with 0.4% Triton X-100 in PBS for 15 minutes to allow antibody access to intracellular targets. Non-specific binding sites were blocked by incubating cells with 10% normal goat serum in PBS for 1 hour at room temperature. Primary antibodies were diluted in blocking solution and applied to cells overnight at 4℃. The next day, cells were washed three times with PBS to remove unbound primary antibodies and incubated with species-specific Alexa Fluor-conjugated secondary antibodies (1:2000) for 1 hour at room temperature. Nuclei were counterstained with 4\u0026rsquo;,6-diamidino-2-phenylindole (DAPI, Beyotime, C1002) for 10 minutes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunofluorescence staining of brain tissue sections\u003c/b\u003e: Brains were isolated and post-fixed in 4% (w/v) paraformaldehyde overnight at 4℃, followed by cryoprotection in 30% sucrose in PBS for 72 hours at 4℃. Coronal brain sections (20 \u0026micro;m thick) were collected using a sliding microtome (Leica). Sections were blocked with 10% normal goat serum in PBS for 1 hour at room temperature and incubated with primary antibodies overnight at 4℃. Primary antibodies included GFP (Rabbit; 1:200; Invitrogen), NeuN (Mouse; 1:200; Abcam), GFAP (Mouse; 1:200; Invitrogen) and MAP2 (Mouse; 1:200; Abcam). After washing three times with PBS, sections were incubated with corresponding Alexa Fluor 488-, 555-, or 647-conjugated secondary antibodies (Invitrogen; 1:1000) for 1 hour at room temperature. Images were captured using a Zeiss LSM700 or Nikon A1R confocal microscope for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDetection of dopamine (DA) in cell culture medium\u003c/h2\u003e \u003cp\u003eDA levels in the supernatant were quantified using a competitive ELISA kit (Elabscience, EEL144) following the manufacturer\u0026rsquo;s protocol. Upon completion of the 28\u0026ndash;30 days culture period of PT109B-induced cells, the culture medium was collected and centrifuged at 1000 \u0026times; g for 20 minutes at 4\u0026deg;C to remove cell debris and impurities. Briefly, a 96-well plate was pre-coated with dopamine antigens. DA present in the samples or standards competed with the immobilized antigens for binding sites on biotin-conjugated anti-dopamine antibodies. Unbound components were removed through washing steps. Horseradish peroxidase-labeled avidin was added, which binds specifically to the biotin-labeled antibodies, forming an immune complex. After another wash step, the substrate 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine was added. 3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine was enzymatically converted to a blue product by Horseradish peroxidase, which turned yellow upon addition of the stop solution (1 M sulfuric acid). Optical density was measured at 450 nm using a microplate reader. The concentration of DA in the samples was inversely proportional to the OD450 values and was determined by interpolation from a standard curve generated using known concentrations of DA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBrdU method for cell proliferation detection\u003c/h2\u003e \u003cp\u003eA BrdU working solution (10 \u0026micro;M) was prepared using freshly pre-warmed cell culture medium. Cells were subsequently treated with neuronal induction medium supplemented with PT109B (10 \u0026micro;M) for 3 or 7 days. The BrdU working solution was added to cultured cells and incubated at 37℃ for 4 hours to allow BrdU incorporation into proliferating cells\u0026rsquo; DNA. Following incorporation, the BrdU-containing medium was replaced with normal culture medium, and cells were further cultured for 24 hours. Cells were washed briefly with PBS and fixed in chilled 70% ethanol for 1 minute, followed by fixation in 4% paraformaldehyde at room temperature for 5 minutes. The fixative was removed, and cells were washed three times with PBS (5 minutes each). To denature DNA and expose BrdU epitopes, cells were treated with 1.5 M HCl at room temperature for 30 minutes. After HCl treatment, cells were washed twice with PBS (5 minutes each). BrdU incorporation was detected using standard immunofluorescence methods. Primary antibodies against BrdU were applied, followed by species-specific secondary antibodies conjugated to fluorescent dyes. Nuclei were counterstained with DAPI, and images were acquired using Nikon fluorescence microscope (Nikon, Eclipse Ti2-E).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCells were lysed using RIPA lysis buffer (containing protease inhibitors), and the resulting lysates were centrifuged to remove cellular debris. Protein concentration was determined using a bicinchoninic acid (BCA) Protein Assay Kit (ThermoFisher, 23225) following the manufacturer\u0026rsquo;s instructions. Based on protein concentration quantified by BCA, 30 \u0026micro;g of protein per well was loaded onto the gel. Electrophoresis was performed at a constant voltage of 80\u0026ndash;120V. The membrane was blocked with 5% bovine serum albumin or 5% non-fat milk in Tris-buffered saline with 0.1% Tween-20 for 2\u0026ndash;3 hours at room temperature to prevent non-specific binding. The blocked membrane was incubated with the primary antibody at 4\u0026deg;C overnight and secondary antibody at room temperature for 1 hour. Protein bands were visualized using a cold charge-coupled device imaging system after exposure to enhanced chemiluminescence substrate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection experiment\u003c/h2\u003e \u003cp\u003e293T cells or primary astrocytes were seeded at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well in six-well plates pre-coated with poly-L-lysine. Transfection was performed using Lipofectamine 2000 (11668500, Invitrogen) according to the manufacturer\u0026rsquo;s protocol. Briefly, plasmid DNA (1 \u0026micro;g) or siRNA (50 nM, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) was diluted in 200 \u0026micro;L of DMEM (Solution A), while 10 \u0026micro;L of Lipofectamine 2000 was diluted in another 200 \u0026micro;L of DMEM (Solution B). Solutions A and B were gently mixed and incubated at room temperature for 10 minutes. Subsequently, Solution B was added to Solution A, gently mixed, and incubated for an additional 20 minutes at room temperature to allow complex formation. The transfection reagent mixture was added dropwise to each well, and cells were incubated 24 hours at 37\u0026deg;C in a 5% CO₂ atmosphere. And the transfection medium was replaced with complete culture medium, and cells were cultured for an additional 24 hours. Following this, the medium was replaced with either regular medium or neuronal induction medium supplemented with PT109B (10 \u0026micro;M) for another 24 hours. Cell morphology was assessed under a phase-contrast microscope to monitor transfection efficiency and overall cell health. Cells were then harvested, and protein lysates were collected for subsequent biomarker analysis using Western blotting.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eFluorescent quantitative PCR reaction\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from cells using TRIzol reagent (Invitrogen, 15596018CN) following the manufacturer\u0026rsquo;s instructions. RNA concentration and purity were assessed using a spectrophotometer. Subsequently, cDNA was synthesized from 1 \u0026micro;g of total RNA using the HiFiScript gDNA Removal cDNA Synthesis Kit (CWBIO, CW2582M), following the provided protocol. qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Q121) on a StepOne Plus Real-Time PCR System (Applied Biosystems). Primer sequences are shown in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The mRNA expression of target genes was normalized to GAPDH and calculated using the 2\u003csup\u003e-△△Ct\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eAntibodies\u003c/h2\u003e \u003cp\u003eGFAP (ab68428), S100β (ab52642), NeuN (ab104224), MAP2 (ab254143), Tyrosine Hydroxylase (TH) (ab75875), Dopamine Transporter (DAT) (ab184451), Ascl1 (ab74065), Brdu (ab1893), OCT4 (ab271937), SOX2 (ab79351), Synaptophysin (ab32127), C3 (ab200999), Dlx2 (ab272902), Islet 1 (lsl1) (ab178400), Lhx8 (ab137036), DDDDK tag (Binds to FLAG tag sequence) (ab205606), Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Antibody, Alexa Fluor 488 (ab150077), Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Antibody, Alexa Fluor 594 (ab150116), Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) Antibody, Alexa Fluor 647 (ab150079), Anti-Mouse IgG (H\u0026thinsp;+\u0026thinsp;L) Antibody, Alexa Fluor 488 (ab150113) were obtained from Abcam.Iba-1 (019\u0026ndash;19741) was obtained from Wako.MAP2 (4542S), DCX (4604s), Ki67 (9129S), NeuroD1 (7019S), Ngn2 (13144S), Tuj1 (5568S), COX2 (12282s), c-Myc (13987S), Brn2 (12137s) were obtained from Cell Signaling Technology.MOG (A5353), Synap1 (A6344), PTBP1 (A6107), TGF-β1 (A25313) were obtained from Abclonal.VGluT1 (48-2400), GFP (A-11122), GAD67 (PA5 21397), Cholera toxin subunit B (recombinant), Alexa Fluor\u0026trade; 488 (C34775), Anti-Sheep IgG (H\u0026thinsp;+\u0026thinsp;L) Antibody, Alexa Fluor 568 (A21099) were obtained from Invitrogen.Nurr1 (10975-2), PKM1 (15821-1-AP), REST (22242-1- AP) were obtained from Proteintech.NaV (ASC-003) was obtained from Alomone.GFAP (MAB360) was obtained from Milipore.GAPDH (AC001) was obtained from ABclonal.Flag (F3165) and HA (H9658) antibodies were obtained from Sigma - Aldrich.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical significance was determined by Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test (for two groups) or one-way or two-way ANOVA, followed by Tukey\u0026rsquo;s multiple comparisons test in using GraphPad Prism 10.4. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant (indicated by an asterisk in the figures). All data were reported as the mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003ePT109B rapidly reprograms astrocytes into induced neurons\u003c/h2\u003e \u003cp\u003eTo examine the impact of PT109B on astrocytes into neuron conversion, we systematically evaluated the temporal dynamics of PT109B mediated astrocytes fate modulation derived from midbrain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Initial characterization confirmed the high purity (\u0026gt;95%) of the primary rat midbrain astrocytes cultures, which exhibited robust immunopositivity for astrocytic markers (GFAP, S100β) while lacking neuronal (NeuN, DCX), microglial (Iba1), and oligodendroglial (MOG) contaminants (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea-c). Firstly, following PT109B treatment (10 \u0026micro;M), we observed a striking temporal progression of cellular transformation. Within 24 hours, astrocytes exhibited significant morphological alterations characterized by reduced cell body size and enhanced process extension, and these changes were much more evident with longer exposure to PT109B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-f). By 7 days post-treatment of PT109B, we detected substantial molecular change, evidenced by decreased GFAP expression accompanied by upregulation of mature neuronal markers (MAP2, NeuN) and dopaminergic-specific proteins (TH, DAT), in time dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). Moreover, the augmented reactivity of activated astrocytes plays a pivotal role in driving their transformation into neurons.\u003csup\u003e15\u003c/sup\u003e PT109B triggered a pattern of astrocytic activation, with immediate (24 hours) upregulation of COX2, TNF-α, and TGF-β1, followed by sustained augmentation of C3, TNF-α, and TGF-β1 levels at 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei, j).\u003c/p\u003e \u003cp\u003eIn the aforementioned results, we observed that PT109B induced astrocytes activation and enhanced the levels of neuron markers as early as 1 day, suggesting that the reprogramming effects of PT109B might be initiated within 24 hours. We further examined that astrocytes reprogramming could be initiated after treatment with PT109B for 0, 0.75, 1.5, 3, 6, 12, 24, and 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We observed that PT109B initiates cellular transformation even earlier, with significant morphological changes occurred within 0.75 hour of treatment and becoming more neuron-like over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). Notably, after 3 hours of PT109B treatment, the protein expression level of Tuj1, an immature neuron marker, exhibited significant differences. As the PT109B treatment duration extended, the expressions of both Tuj1 and DCX were significantly elevated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el, m), and the number of Tuj1\u003csup\u003e+\u003c/sup\u003e cells was significantly higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003en, o). Together, these results indicate that PT109B is a remarkably rapid and potent inducer of astrocytes-to-neuron reprogramming.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003ePT109B reprograms midbrain astrocytes into functional iDANs\u003c/h2\u003e \u003cp\u003eGiven that PT109B can rapidly reprogram astrocytes into induced neurons expressing with DAN-specific markers, we further evaluated its potential to generate mature and functional iDANs. After PT109B treatment for half a month, we observed that the emergence of MAP2\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and TH\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) from the astrocyte population, with these cells exhibiting neuronal morphology. Quantitatively, PT109B treatment resulted in approximately 70\u0026ndash;85% MAP2\u003csup\u003e+\u003c/sup\u003e and NeuN\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), with 20% of them expressing tyrosine hydroxylase (TH), a hallmark of dopaminergic identity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). When the PT109B treatment was extended to 1 month, the PT109B-induced cells also demonstrated a significant expression of another dopaminergic markers DAT and Nurr1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, d), and synaptic protein Synap1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). And a significant increase in NaV\u003csup\u003e+\u003c/sup\u003e cells was observed in astrocytes, with NaV channel signals primarily localized to the cell bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Furthermore, we observed significantly elevated dopamine levels in the medium cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei), collectively confirming the acquisition of dopaminergic neuron properties.\u003c/p\u003e \u003cp\u003eTo confirm the electrophysiological maturity, we started to test the capability of action potential generation from the 1-month PT109B-treated cells. The PT109B-treated cells not only progressively acquired morphological characteristics of neurons from 1- to 3-month treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). The 1 to 3-month post-PT109B treatment cells displayed a rudimentary depolarized peak (half-width\u0026thinsp;\u0026gt;\u0026thinsp;5 ms, amplitude\u0026thinsp;\u0026gt;\u0026thinsp;50 mV) with a significant reduction in capacitance and augmentation in peak amplitude, accounting for more than 40% of recorded neuron-like cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, j-l). However, these cells did not demonstrate a well-developed inward current, and no typical action potential (AP) was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eTo further enhance iDANs electrophysiological functions, we supplemented PT109B with 50 nM thyroid hormone T3 for half a month. T3 plays an important role in regulating neuronal excitability and enhancing synaptic efficacy through its action on Na-K-ATPase activity, which modulates ion homeostasis and membrane potential[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The combination of treatment yielded more than 45% of recorded neuron-like cells exhibiting AP, particularly 15 cells generating multiple APs within 1 month (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em). PT109B-treated cells, especially with T3, showed significantly increased input resistance along with mature neuronal morphological and electrophysiological properties, including fine and elongated dendrite/axon-like processes, capability for continuous firing, voltage-dependent sodium and potassium currents, and spontaneous postsynaptic currents (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en-r). These induced properties indicate the enhanced neuronal functions and potential of neuronal network formation. Collectively, these results demonstrate that PT109B alone suffices to generate iDANs with basic structural and functional properties, while its combination with T3 produces relatively mature electrophysiological functions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003ePT109B directly reprograms astrocytes into iDANs without involving a neural progenitor stage\u003c/h2\u003e \u003cp\u003eIt is reported that pluripotent stem cells could be generated by chemical reprogramming. The potential tumor risks of pluripotent stem cell may hamper their usage[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Thus, direct lineage reprogramming astrocytes by small molecular combinations provides a valuable approach for generating neurons. To address whether the reprogramming process by PT109B involving a stem cell-like intermediate stage, we systematically analyzed the expression of neural stem cell markers (SOX2, OCT4) and an immature neuronal marker (Tuj1) in astrocytes, treated with PT109B, for 3 and 7 days (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea). Strikingly, we observed a significant increase in the number of Tuj1\u003csup\u003e+\u003c/sup\u003e cells in PT109B-treated astrocytes compared to the control group, whereas SOX2\u003csup\u003e+\u003c/sup\u003e and OCT4\u003csup\u003e+\u003c/sup\u003e cells were entirely absent (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb-d). Given the robust proliferative capacity of neural stem cells, we also evaluated cell proliferation during PT109B reprogramming. Astrocytes were pre-labeled with BrdU before PT109B treatment, and the presence of BrdU\u003csup\u003e+\u003c/sup\u003e and Ki67\u003csup\u003e+\u003c/sup\u003e cells was assessed on days 3 and 7 post-treatment. Notably, the number of BrdU\u003csup\u003e+\u003c/sup\u003e and Ki67\u003csup\u003e+\u003c/sup\u003e cells in PT109B-treated groups was significantly reduced compared to the control (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ee-g). These findings collectively confirm that PT109B-mediated reprogramming bypasses a stem cell-like stage and concurrently suppresses cell proliferation.\u003c/p\u003e \u003cp\u003eFurther supporting this notion, we observed progressive morphological changes in astrocytes being treated with PT109B over 3, 7, and 14 days. The treated cells exhibited neuronal characteristics, including smaller cell bodies and increased length and branching of processes. Concurrently, the number of GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes decreased, while the cells co-expressing GFAP, MAP2, and TH markers emerged (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eh). Statistical analysis revealed an inverse correlation between the number of GFAP\u003csup\u003e+\u003c/sup\u003e cells and the duration of PT109B treatment, whereas the proportions of MAP2\u003csup\u003e+\u003c/sup\u003e and TH\u003csup\u003e+\u003c/sup\u003e cells positively correlated with the treatment duration (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ei-k). Taken together, these results suggest that PT109B directly reprograms midbrain astrocytes into functional iDANs without transitioning through a stem cell-like intermediate.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePT109B reprograms astrocytes into neurons by altering multiple transcription factors\u003c/h3\u003e\n\u003cp\u003eBasic helix-loop-helix (bHLH) factors, such as Ascl1 (also called Mash1), NeuroD1, and Neurogenin 2 (Ngn2), play a key role in regulating the fate determination and reprogramming of astrocytes into neurons[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have demonstrated that overexpression of transcription factors Ascl1 and NeuroD1 can reprogram astrocytes into iDANs[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. To elucidate the underlying molecular mechanisms of PT109B-mediated reprogramming, we employed fluorescent quantitative PCR to analyze the mRNA expression levels of key neuronal markers and transcription factors in astrocytes following 3 days of PT109B treatment. Notably, PT109B significantly upregulated the mRNA levels of neuronal markers, including MAP2, NeuN, Tuj1, and DCX (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), as well as transcription factors such as NeuroD1, Ngn2, Ascl1, Nurr1, and Dlx2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Furthermore, PT109B also enhanced the mRNA expressions of MAP2, TH and NeuroD1 in astrocytes following 7 days of PT109B treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Then, we exposed astrocytes to PT109B for varying durations (0, 3, 6, 12, 24, 72, and 168 hours) and analyzed the expression of key bHLH transcription factor. Consistent with our previous observations, the mature neuronal marker MAP2 began to increase after 3 days of PT109B treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). Notably, within 12 hours of exposure, we observed a significant upregulation of Ascl1, Ngn2, and NeuroD1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, e). Concurrently, levels of c-Myc, PTBP1, and REST, known inhibitors of neuronal differentiation, were markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). In contrast, transcription factors associated with stem cell maintenance (Isl-1, SOX2, OCT4, and Brn2) and cholinergic neuronal differentiation (Lhx8) were largely unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, g). These results suggest that PT109B specifically targets a subset of transcription factors critical for neuronal reprogramming, including c-Myc, Ascl1, Ngn2, Nurr1, NeuroD1, and Dlx2.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that suppression of PTBP1 enhances the expression of NeuroD1, Ascl1, and neuronal-related genes in astrocytes[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Interestingly, our results revealed that PT109B significantly reduces PTBP1 levels (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ea, b), implicating PTBP1 as a potential mediator of PT109B-induced reprogramming. To further explore this, we compared the effects of PT109B treatment and PTBP1 knockdown in astrocytes. While both interventions reduced PTBP1 levels, only PT109B induced significant morphological changes characteristic of neuronal reprogramming (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ec, d). Moreover, even under conditions of PTBP1 overexpression, PT109B successfully reduced PTBP1 level and initiated morphological transformation (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003ee, f). These findings demonstrate that PT109B orchestrates astrocytes-to-dopaminergic neuron conversion through the coordinated regulation of bHLH transcription factors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptomic changes during PT109B initiation of astrocytes reprogramming\u003c/h2\u003e \u003cp\u003eBuilding on our earlier discovery that PT109B initiates astrocytes reprogramming within 3\u0026ndash;6 hours, we sought to unravel the underlying molecular mechanisms driving this process. We conducted transcriptomic analysis at 1.5, 3, and 6 hours post-PT109B treatment. Principal component analysis (PCA) revealed a progressive divergence between PT109B-treated and control groups, indicating substantial transcriptional rewiring over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b). Differential gene expression analysis identified a significant number of genes with fold changes\u0026thinsp;\u0026gt;\u0026thinsp;2 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 in each group. Specifically, PT109B treatment resulted in the upregulation of 647 genes and downregulation of 1179 genes at 1.5 hours, 557 upregulated and 2129 downregulated genes at 3 hours, and 707 upregulated and 1913 downregulated genes at 6 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Functional annotation of these differentially expressed genes (DEGs) revealed a marked decline in pathways associated with cell cycle, RNA splicing, DNA binding, and glial cell proliferation and differentiation. Conversely, genes involved in neuronal differentiation, neurogenesis, dopamine neuron differentiation, and extracellular matrix organization exhibited gradual upregulation over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). KEGG pathway analysis further highlighted significant enrichment in cell cycle regulation, TNF inflammatory signaling, TGF-β signaling, and neural regeneration pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). These findings suggest that PT109B-mediated reprogramming rapidly inhibits cell cycle progression and activates inflammation-related pathways, while simultaneously promoting neurogenesis-associated gene expressions.\u003c/p\u003e \u003cp\u003eTo identify key regulatory genes driving these early transcriptional changes, we analyzed DEGs across time points and visualized them in volcano plots, selecting the top ten significantly altered genes for further investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef-h). Pathway impact analysis revealed that these genes primarily suppressed cell cycle and tumor-associated microRNA regulatory pathways while enhancing MAPK and TGF-β signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei). The upregulated DEGs were predominantly associated with neurodegeneration, inflammation, DNA binding, and cytoskeletal regulation, while the downregulated DEGs were enriched in cell cycle progression, proliferation, and lipid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej). Gene network analysis within these pathways identified critical roles for transcription factor-associated proteins, including Smad3, Fosb, Sp1, and Myc, whereas PTBP1 and PTBP2 showed limited interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek). To validate the RNA sequencing results, we employed qPCR to evaluate the expression profiles of critical signaling pathways following 6 hours of PT109B-induced reprogramming. The mRNA levels of genes involved in the TGF-β/BMP signaling pathway (TGF-β1, BMP1, SMAD3), the MAPK signaling pathway (MAPK1, Fosb, Myc, Ptgs2) were significantly upregulated (Fig. S4a, b). In contrast, the mRNA levels of cell cycle regulation (Mki67, Pten) and PTBP1 were markedly downregulated (Fig. S4a-c). These results collectively demonstrate that PT109B orchestrates early transcriptional reprogramming by modulating DNA- and RNA-related functional proteins, leading to the activation of MAPK and TGF-β signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003ePT109B improves motor function in 6-OHDA-induced PD mice\u003c/h2\u003e \u003cp\u003eTo evaluate PT109B efficacy \u003cem\u003ein vivo\u003c/em\u003e within the context of PD, we employed a 6-OHDA-induced PD mouse model, and selectively targeting astrocytes via adeno-associated virus (AAV5-GFAP-EGFP) delivery to the substantia nigra and striatum (Fig. S5a). Three weeks following 6-OHDA injection into the medial forebrain bundle (MFB), apomorphine-induced rotation tests confirmed successful PD modeling (Fig. S5b). Immunofluorescence analysis revealed a marked reduction in TH\u003csup\u003e+\u003c/sup\u003e neurons in the substantia nigra and striatum of PD mice compared to controls, validating the model\u0026rsquo;s neuropathological fidelity (Fig. S5c). Further, GFP\u003csup\u003e+\u003c/sup\u003e cells were found to co-localize with GFAP but not NeuN, confirming specific targeting of astrocytes rather than neurons (Fig. S5d, e).\u003c/p\u003e \u003cp\u003eTo assess the therapeutic potential of PT109B, PD mice were orally administered PT109B (100 mg/kg) for 12 weeks. At week 18, retrograde labeling was achieved via cholera toxin B (CTB) injection into the striatum, followed by behavioral and pathological assessments 1 week later (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Apomorphine-induced rotation tests demonstrated a significant reduction in rotational behavior in PT109B-treated PD mice compared to untreated PD controls, while selegiline (a standard PD treatment) showed only a moderate, non-significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Notably, PT109B administration in normal mice had no discernible impact on behavior. Motor function improvements were further evidenced by the pole climbing assay, where PT109B-treated PD mice exhibited substantially reduced climbing times compared to untreated PD mice, with selegiline-treated mice showing a lesser improvement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The rotarod test showed that in PD mice treated with PT109B, the motor coordination ability was enhanced and the endurance time increased, but there was no statistically significant difference found (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). The hindlimb clasping test further corroborated these findings, with PT109B significantly ameliorating limb posture in PD mice, as reflected by reduced flexion-extension scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Exploration of anxiety-related behaviors via open field tests revealed that PT109B and selegiline treatments increased central area exploration and movement velocity in PD mice, suggesting slight improvements in both motor function and anxiety (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-i). Collectively, these results demonstrate that PT109B effectively restores motor function and ameliorates the PD-associated behavioral deficits \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePT109B increases the number of DANs in the brain of PD mice and reprogrammed astrocytes into induced neurons with electrophysiological activity\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the therapeutic potential of PT109B, we investigated its effects on DAN density and neural circuitry restoration in the substantia nigra and striatum of 6-OHDA-induced PD mice. DANs in the damaged striatum and substantia nigra of the model group showed significant reduction compared to controls, accompanied by marked morphological degeneration, including loss of intact neuronal soma (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). In contrast, the PT109B-treated mice exhibited a pronounced increase in DAN density in these regions, with preserved neuronal morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b). Immunofluorescence analysis further demonstrated elevated levels of NeuN\u003csup\u003e+\u003c/sup\u003e and TH\u003csup\u003e+\u003c/sup\u003e cells in PT109B-treated mice, indicating not only enhanced DAN numbers but also the restoration of neuronal integrity. In comparison, selegiline treatment failed to significantly restore DAN density or improve neuronal morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-e).\u003c/p\u003e \u003cp\u003eTo assess whether PT109B could reprogram astrocytes into functional neurons \u003cem\u003ein vivo\u003c/em\u003e, we injected AAV5-GFAP-EGFP into the striatum and substantia nigra to label astrocytes, followed by oral administration of PT109B (100 mg/kg) for 3 months. While GFP\u003csup\u003e+\u003c/sup\u003e cells with neuron-like morphology were observed, no significant co-labeling of GFP\u003csup\u003e+\u003c/sup\u003e and NeuN\u003csup\u003e+\u003c/sup\u003e cells was detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), suggesting incomplete reprogramming of astrocytes into mature neurons. To evaluate the restoration of dopaminergic circuitry, we injected the retrograde tracer CTB into the striatum. Control mice exhibited robust CTB and TH co-labeling in the substantia nigra, indicative of intact dopaminergic projections. In contrast, PD model mice showed diminished CTB signals and significant DAN loss. Remarkably, PT109B treatment restored both CTB signals and DAN density, with co-labeling of CTB and TH, suggesting the structural recovery of dopaminergic projections (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, h). Electrophysiological recording of GFP-labeled cells in the striatum revealed two distinct populations: (i) cells with very small cell bodies lacking APs and sodium-potassium currents, consistent with astrocytes; and (ii) larger and rounded cells exhibiting neuronal activity are capable to generate repetitive APs and show large voltage-gated sodium and potassium currents (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei-l). These findings suggest that PT109B may partially induce neuronal-like properties in astrocytes without achieving full neuronal conversion.\u003c/p\u003e \u003cp\u003eTo assess the safety of long-term PT109B administration, we conducted chronic toxicity studies in both C57BL/6 and PD mice. Oral administration of PT109B (100 mg/kg) for 3 months did not affect body weight or organ indices (heart, liver, spleen, lungs, kidneys, and brain) in any group (Fig. S6a, b). Histopathological examination (H\u0026amp;E staining) revealed no evidence of organ damage, confirming the absence of pathological changes (Fig. S6c, d). Liver function assays indicated no significant alterations in alanine aminotransferase (ALT), aspartate aminotransferase (AST), or gamma-glutamyl transferase (γ-GT) levels (Fig. S6e-g). Similarly, kidney function markers, including blood urea nitrogen (BUN), creatinine (CREA), and uric acid (UA), remained unchanged across all groups (Fig. S6h-j). Hematological analysis revealed no significant differences in white blood cells, neutrophil, lymphocytes, or monocyte counts (Fig. S6k-n). Red blood cell parameters, including mean corpuscular volume, mean corpuscular hemoglobin, and red blood cell count, were unaffected by PT109B (Fig. S6o-q). While hemoglobin levels were slightly elevated in PT109B-treated mice, these changes were not statistically significant compared to controls (Fig. S6r). Platelet count and mean platelet volume also remained unaltered (Fig. S6s, t). Together, these results demonstrate that PT109B exhibits an excellent safety profile, supporting its potential for long-term therapeutic use in PD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified PT109B as a potent inducer of reprogramming astrocytes into functional iDANs. Remarkably, PT109B initiated astrocytes reprogramming within 3 hours. The reprogrammed iDANs exhibited synaptic structures, neuron-like electrophysiological activity, dopamine release capabilities, and long-term survival (for over 3 months) \u003cem\u003ein vitro\u003c/em\u003e. Importantly, oral administration of PT109B in a 6-OHDA-induced PD mouse model not only alleviated motor deficits but also increased the number of dopaminergic neurons in the striatum and substantia nigra. By overcoming key bottlenecks in chemical reprogramming and demonstrating therapeutic potential in PD models, PT109B provides a promising foundation for developing tools useful for regenerative therapies aiming at for neurodegenerative diseases.\u003c/p\u003e \u003cp\u003eCompared to mature neurons, astrocytes possess a lower resting membrane potential (approximately \u0026minus;\u0026thinsp;90 mV) and reduced membrane resistance. The large surface area and low membrane resistance of astrocytes make the detection of AP inherently challenging[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, the single AP, generated by PT109B-induced cells, were of modest amplitude, with relatively small sodium currents, indicating the presence of functional sodium and potassium channels and the initiation of depolarization. While the degree of depolarization was limited, these changes significantly altered the electrophysiological profile of astrocytes, marking a critical step toward neuronal maturation. The Na-K-ATPase, a membrane protein essential for regulating intracellular and extracellular sodium and potassium ion concentrations, plays a pivotal role in maintaining the osmotic balance and facilitating depolarization and hyperpolarization in cells[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We hypothesize that the inability of PT109B to induce continuous AP may stem from insufficient Na-K-ATPase activity in the reprogrammed cells. Supporting this, T3, a biologically active hormone, has been shown to specifically enhance Na-K-ATPase activity. T3 (50 nM) promotes changes in the extracellular matrix of astrocytes[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. To address the limitations in PT109B-induced electrophysiological activity, we supplemented the induction system with T3 (50 nM). This combination markedly increased the proportion of cells exhibiting continuous AP after 15 or 30 days of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em). Additionally, the amplitude of sodium currents significantly increased in bipolar or tripolar induced neurons with smaller cell bodies, and some cells displayed excitatory postsynaptic electrophysiological properties. These results indicate that T3 synergistically enhances the electrophysiological maturation of PT109B-induced neurons, enabling the detection of continuous AP in treated astrocytes.\u003c/p\u003e \u003cp\u003eNotably, the reprogramming of fibroblasts into induced pluripotent stem cells (iPSCs) using chemical small molecules has been shown to involve cell cycle arrest, a state characterized by paused cell division and delayed development. During this process, histone protein acetylation increases, enhancing chromatin accessibility and promoting reprogramming efficiency. Similarly, modulating cell proliferation, such as by knocking out p53 in somatic cells, including fibroblasts and astrocytes, has been demonstrated to significantly enhance the reprogramming efficiency of transcription factors[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. PT109B has been shown to induce cell cycle arrest in glioma cells, promoting their transformation into neuron-like cell[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. These parallels suggest that the cell cycle arrest state induced by PT109B is a key factor facilitating the direct conversion of astrocytes into neurons. The emergence of intermediate neuron-like cells (MAP2\u003csup\u003e+\u003c/sup\u003e/TH\u003csup\u003e+\u003c/sup\u003e/GFAP\u003csup\u003e+\u003c/sup\u003e) during PT109B-induced reprogramming further supports the notion that astrocytes can be directly converted into neurons. Given the shared lineage and functional similarities between astrocytes and neurons, this direct conversion is biologically plausible[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The appearance of intermediate cells expressing both neuronal and glial markers suggests a transitional state during reprogramming, wherein astrocytes progressively adopt neuronal characteristics while shedding their glial identity.\u003c/p\u003e \u003cp\u003eOur findings reveal that PT109B induces profound morphological changes, upregulates neuronal markers, and enhances cellular activation and oxidative stress levels during the reprogramming process. These effects are mediated, in part, by the upregulation of b-HLH family transcription factors, including Ascl1, Ngn2, and NeuroD1. These factors are known to play critical roles in neuronal differentiation and maturation. Previous studies have demonstrated that the combinations of chemicals can reprogram astrocytes into neurons within 2\u0026ndash;4 days, accompanied by dynamic changes in b-HLH family transcription factors (Ascl1, NeuroD1, and Ngn2)[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Consistent with this, we observed that PT109B significantly elevated the protein levels of these transcription factors within 6 hours, while simultaneously reduced the expressions of inhibitory proteins PTBP1 and REST. This rapid molecular response is accompanied by a marked activation of astrocytes. Although PTBP1 knockdown has been reported to reprogram astrocytes into DANs, recent studies have cast doubt on its role as a gatekeeper gene in this process[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. While we initially hypothesized that PTBP1 might be a key mediator of PT109B's effects, the overexpression of PTBP1 in PT109B-treated astrocytes failed to reverse the morphological changes induced by the compound. However, short-term transcriptomic analysis revealed that the PTBP1 pathway is may not a primary target of PT109B, suggesting that other mechanisms could account its reprogramming efficacy.\u003c/p\u003e \u003cp\u003eOur RNA sequencing data highlights the upregulation of pathways associated with cellular activation, including TGF-β, MAPK, and TNF-α, within 6 hours of PT109B treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek, l). Conversely, the cell cycle pathway is significantly suppressed. These findings align with previous reports that overexpression of b-HLH family transcription factors induces a state of high oxidative stress and cell cycle arrest during neuronal reprogramming[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The MAPK cascade, a central hub for signal transduction, regulates diverse cellular processes, including proliferation, differentiation, and apoptosis. In unstimulated cells, MAPK remains inactive; however, stimulation triggers its activation via MKK and MKKK-mediated phosphorylation[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. PT109B treatment significantly enhances MAPK pathway activity, indicating a highly activated state in treated astrocytes. This activation coincides with elevated expression of neural stem cell genes, and elevated levels of inflammatory factors like TNF-α and TGF-β1[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These changes suggest that astrocytes acquire a stem-like state, characterized by an open chromatin conformation that facilitates gene expression. These findings suggest that PT109B-induced TNF-α elevation plays a pivotal role in astrocytes activation and dedifferentiation, warranting further investigation into the precise signaling mechanisms involved. Interestingly, PT109B also significantly increases TGF-β1 levels, alongside elevated BMP1 and SMAD3 expression. TGF-β1, primarily sequestered in the extracellular matrix, is released in response to extracellular oxygen radicals or integrin signaling. Upon binding to the TGF-βR1/2 receptor complex, it triggers SMAD2/3 phosphorylation and subsequent nuclear translocation of SMAD2/3-SMAD4 complexes, regulating genes involved in cell activation and differentiation[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Whether the TGF-β pathway is a key mediator of PT109B's effects remains to be explored.\u003c/p\u003e \u003cp\u003eEpigenetic reprogramming mechanism is likely enhanced by small chemical molecules, which promote DNA accessibility and transcriptional activation[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Recent advancements in epigenomic profiling, including ATAC-seq, histone modification omics, and DNA methylation analyses, have identified key targets of small molecule-mediated reprogramming[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Applying these techniques in future studies could elucidate the epigenetic changes driving PT109B-induced astrocytes reprogramming and identify its molecular targets.\u003c/p\u003e \u003cp\u003eTo further evaluate PT109B therapeutic potential for PD \u003cem\u003ein vivo\u003c/em\u003e, we employed the 6-OHDA-induced PD mouse model, which exhibits high DAN mortality, and irreversible PD symptoms[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Our results demonstrate that PT109B administration significantly ameliorates PD-associated behavioral deficits, including anxiety-like behavior, reduced motor speed, endurance, and impaired coordination. Furthermore, PT109B increased the number of DANs in the lesioned striatum of PD mice. To investigate the fate of astrocytes \u003cem\u003ein vivo\u003c/em\u003e, we utilized AAV5-GFAP-EGFP to specifically label astrocytes in the brains of PD mice. While GFP\u003csup\u003e+\u003c/sup\u003e cells exhibited morphological and electrophysiological characteristics resembling neurons, we did not observe co-localization of GFP with the mature neuronal marker NeuN (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef). This suggests that PT109B may not induce fully reprogram of astrocytes into mature neurons but rather it may induce an immature neuron-like state. To test this hypothesis, future studies could assess the co-expression of GFP with immature neuronal markers such as DCX. However, limitations associated with AAV-based tracing, including potential leakage and loss of fluorescence labeling and the influence of transcription factor levels on GFAP promoter activity, underscore the need for more reliable lineage-tracing tools[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Future studies utilizing astrocytes lineage tracing system, such as Aldh1l1-CreERT2 mice may provide more definitive insights into the \u003cem\u003ein vivo\u003c/em\u003e reprogramming effects of PT109B. Additionally, we employed retrograde tracing with CTB injected into the striatum to monitor dopaminergic projections. Compared to the control group, PT109B-treated PD mice exhibited significant CTB\u003csup\u003e+\u003c/sup\u003e/TH\u003csup\u003e+\u003c/sup\u003e co-staining in the substantia nigra, indicating partial recovery of the nigrostriatal dopaminergic pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, h). This finding is consistent with the observed behavioral improvements and increased DAN counts, further supporting the neurorestorative potential of PT109B.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTogether, PT109B is shown to be a promising small molecule that can reprogram astrocytes into functional iDANs, alleviate PD-related motor deficits and contribute to the restoration of dopaminergic neuron populations, presenting an alternative option for the treatment of PD.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"99%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003ePD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eParkinson\u0026apos;s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eiDANs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eInduced dopaminergic neurons\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eb-HLH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eBasic Helix-Loop-Helix\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eDANs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eDopaminergic neurons\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003e6-OHDA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e6-hydroxydopamine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eHP-\u0026beta;-CD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eHydroxypropyl-\u0026beta;-cyclodextrin\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eAnterior-posterior\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eML\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eMedio-lateral\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eDV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eDorso-ventral\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eCTB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eCholera toxin B\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eACSF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eArtificial Cerebrospinal Fluid\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eDEGs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eDifferentially expressed genes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eGO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eGene ontology\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eSprague-Dawley\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003ePBS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eDAPI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e4\u0026rsquo;,6-diamidino-2-phenylindole\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eTMB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e3,3\u0026rsquo;,5,5\u0026rsquo;-tetramethylbenzidine\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003ePCA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003ePrincipal component analysis\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 26px;\"\u003e\n \u003cp\u003eMFB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eMedial forebrain bundle\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to Prof. Zhongwei Zhou for generously providing the mouse primary neural stem cells. Our appreciation also goes to the Guangdong Provincial Key Laboratory of Brain Function and Disease for their invaluable guidance and support in confocal microscopy. We are deeply thankful to Prof. Gao Jin and Dr. Leung Ka Wing from the Division of Life Science and the Center for Chinese Medicine at The Hong Kong University of Science and Technology. Their expertise and assistance were instrumental in the design and execution of the animal experiments. Graphical abstract was created by Biorender.com.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.Y., C.L.W., and Z.Y.Z designed the overarching concepts for the study. T.H.F., J.W.Z., and Y.L.T planned the research methods. S.S.W. developed the necessary software tools. C.L.W., Y.L., D.C., and Z.X.Z. carried out validation processes. Y.Y., C.L.W., and Y.R. conducted formal analyses. C.L.W., Y.Y., K.J., and S.Q.H. were involved in research investigations. W.B.D., Q.Z., and C.L. provided the required resources. Y.L. and Y.P.C. curated the research data. C.L.W., Y.Y., and T.H.F. designed experiments for the initial draft and wrote it. R.B.P. and K.W-K.T. helped refine the manuscript with their review and editing work. Y.Y., C.L.W., S.S.W., and Z.L. designed visualizations and helped present the data. R.B.P. supervised the overall research process. R.B.P. and K.W-K.T. managed the project. R.B.P. acquired the necessary funding for the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Shenzhen Science and Techonlogy Program (Grant No. 202111233000079) to R.B.P.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll RNA-seq raw data and count files were deposited in the Gene Expression Omnibus as a Super Series under accession number GEO: GSE292869. Raw western blot data are deposited in Mendeley Data (DOI:10.17632/gmd3t57fnt.1) Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments conducted were approved by the Institutional Animal Care and Use Committee (IACUC) of Sun Yat-sen University, and were performed under SYSU-IACUC-2021-B0137. Clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that there are no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eSchool of Medicine, Sun Yat-sen University - Shenzhen Campus, Shenzhen 518107, China. \u003csup\u003e2\u003c/sup\u003eSchool of Pharmacy/Key Laboratory of Xinjiang Phytomedicine Resource and Utilization Ministry of Education/Institute for Safflower Industry Research, Shihezi University, Shihezi 832000, China. \u003csup\u003e3\u003c/sup\u003eSchool of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China. \u003csup\u003e4\u003c/sup\u003eSchool of Life Sciences, South China Normal University, Guangzhou 510631, China. \u003csup\u003e5\u003c/sup\u003eSchool of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China. \u003csup\u003e6\u003c/sup\u003eSchool of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong 999077, China. \u003csup\u003e7\u003c/sup\u003eSchool of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University - Shenzhen Campus, Shenzhen 518107, China. \u003csup\u003e8\u003c/sup\u003eDivision of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong 999077, China. \u003csup\u003e9\u003c/sup\u003eInternational Joint Laboratory (SYSU-PolyU HK) of Novel Anti-Dementia Drugs of Guangdong, Guangzhou 510006, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTanner CM, Ostrem JL. 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Cell.\u003cem\u003e \u003c/em\u003e2021; 184(21):5465-5481.e16.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Astrocytes, Reprogramming, Induced dopaminergic neurons, PT109B, Parkinson’s disease","lastPublishedDoi":"10.21203/rs.3.rs-6428230/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6428230/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eParkinson\u0026rsquo;s disease (PD) is characterized by the progressive loss of dopaminergic neurons (DANs), leading to motor dysfunction, while current treatments fail to restore neuronal loss. Reprogramming astrocytes into induced DANs by small molecules offers a promising therapeutic strategy, but existing methods face challenges including low efficiency and complex mechanisms. PT109B, a novel multi-kinase inhibitor, has demonstrated neurogenic and synaptogenic potential in neural progenitor cells, as well as glioblastoma differentiation capacity, yet its ability to directly convert astrocytes into functional DANs and its therapeutic effects in PD remain unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePrimary rat midbrain astrocytes were treated with 10 \u0026micro;M PT109B to evaluate reprogramming efficiency via immunofluorescence (GFAP, MAP2, NeuN, TH, DAT) and electrophysiological recordings. RNA sequencing was performed at 1.5, 3, and 6 hours post-treatment to assess transcriptional changes. In vivo, PT109B (100 mg/kg) was administered orally for 12 weeks in 6-OHDA-induced PD mice, with astrocytes labeled by AAV5-GFAP-EGFP. Behavioral tests (apomorphine rotation, pole test, rotarod, and open field), retrograde tracing, and immunohistochemistry were conducted to evaluate therapeutic effects.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003ePT109B initiated astrocyte-to-neuron conversion as early as 3 hours, yielding 20% TH⁺ dopaminergic neurons by 2 weeks in vitro, with mature electrophysiological properties for action potentials, sodium currents and sustained dopamine release (\u0026gt;\u0026thinsp;3 months). Mechanistically, PT109B drove this conversion through cell cycle arrest, astrocytic activation, and upregulation of key basic Helix-Loop-Helix (b-HLH) transcription factors (NeuroD1, Ascl1, Ngn2). \u003cem\u003eIn vivo\u003c/em\u003e, oral administration of PT109B in a 6-OHDA-induced PD mouse model exhibited significant therapeutic efficacy by reprogramming astrocytes to functional neurons in the striatum, leading to improved motor functions.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003ePT109B efficiently converts astrocytes into functional induced DANs through rapid reprogramming and ameliorates PD-related pathology and motor deficits, presenting a safe and effective single-molecule therapeutic strategy for PD.\u003c/p\u003e","manuscriptTitle":"PT109B, a Multikinase Inhibitor, Converts Astrocytes into Dopaminergic Neurons and Alleviates Parkinson's Disease in Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-25 10:11:57","doi":"10.21203/rs.3.rs-6428230/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71b6b359-716b-4726-8625-5f2597dbffea","owner":[],"postedDate":"April 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-04T03:35:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-25 10:11:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6428230","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6428230","identity":"rs-6428230","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}