Bridging Cytoskeletal and Epitranscriptomic Mechanisms: L-DOPA–Induced Microtubule Remodeling Meets m⁶A RNA Methylation in Neural Disorders

Bridging Cytoskeletal and Epitranscriptomic Mechanisms: L-DOPA–Induced Microtubule Remodeling Meets m⁶A RNA Methylation in Neural Disorders | 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 Systematic Review Bridging Cytoskeletal and Epitranscriptomic Mechanisms: L-DOPA–Induced Microtubule Remodeling Meets m⁶A RNA Methylation in Neural Disorders Sonu kumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8217923/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 Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuronal loss, α-synuclein aggregation, and disrupted motor and non-motor function (Bloem et al., 2021 ). Pharmacological replenishment of dopamine using L-3,4-dihydroxyphenylalanine (L-Dopa) remains the cornerstone of PD management; however, long-term exposure frequently induces motor fluctuations, dyskinesias, and cognitive side effects (Olanow & Obeso, 2021 ). Recent mechanistic evidence reveals that L-Dopa itself, independent of its metabolic conversion to dopamine, can be aberrantly incorporated into neuronal microtubules, leading to structural and synaptic instability (Zorgniotti et al., 2025 ). L-Dopa acts as a tyrosine analogue within the tubulin tyrosination–detyrosination cycle, where tubulin tyrosine ligase (TTL) catalyzes its attachment to α-tubulin. The resulting L-Dopa–modified microtubules resist enzymatic removal by the vasohibin-1/small vasohibin-binding protein (VASH1–SVBP) complex, thereby accumulating in neurons and impairing cytoskeletal plasticity (Peris & Moutin, 2023 ; Zorgniotti et al., 2025 ). Functionally, this modification disrupts dendritic spine invasion, reduces excitatory synaptic density, and perturbs intracellular transport, culminating in synaptic weakening and neurofunctional decline. These findings introduce a cytoskeletal dimension to L-Dopa neurotoxicity, linking pharmacotherapy to microtubule dysregulation and altered neuronal connectivity. Understanding the molecular interface between L-Dopa metabolism and tubulin dynamics may inform strategies to mitigate long-term treatment complications and preserve synaptic integrity in PD. Parkinson’s disease L-Dopa microtubule dynamics tubulin tyrosination–detyrosination cycle tubulin tyrosine ligase (TTL) vasohibin-1 (VASH1) cytoskeletal remodeling dendritic spine stability synaptic plasticity dopaminergic neurotoxicity motor fluctuations neurodegeneration Figures Figure 1 2. Introduction 2.1. Parkinson’s Disease and the Central Role of L-Dopa Parkinson’s disease (PD) represents the second most prevalent neurodegenerative disorder, affecting approximately 1–2% of individuals over 65 years of age worldwide (Bloem et al., 2021 ). The pathological hallmark of PD is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to striatal dopamine depletion and motor deficits such as bradykinesia, rigidity, and tremor (Kalia & Lang, 2015 ). Since its clinical introduction in 1967, L-Dopa has remained the most effective symptomatic therapy, restoring dopaminergic transmission and improving motor performance (Fahn, 2015 ). Nevertheless, chronic administration often results in L-Dopa-induced dyskinesia (LID) and fluctuating “on–off” motor responses, implicating maladaptive plasticity within basal ganglia circuits (Olanow & Obeso, 2021 ). Although dopaminergic oxidation and reactive oxygen species generation have been traditionally implicated in L-Dopa toxicity, emerging data suggest that the molecule exerts direct, dopamine-independent effects on neuronal macromolecules, prompting renewed interest in its cellular interactions beyond neurotransmitter metabolism (Zorgniotti et al., 2025 ). 2.2. Microtubule Dynamics and the Tubulin Tyrosination–Detyrosination Cycle Microtubules are dynamic cytoskeletal polymers essential for axonal transport, neuronal polarity, and synaptic plasticity (Kapitein & Hoogenraad, 2015 ). Their post-translational modification landscape, collectively termed the “tubulin code,” regulates motor-protein binding and cytoskeletal remodeling (Janke & Magiera, 2020 ). Among these modifications, the reversible tyrosination–detyrosination cycle of α-tubulin is critical for maintaining microtubule turnover and function. Tubulin tyrosine ligase (TTL) catalyzes the ATP-dependent attachment of L-tyrosine to the C-terminus of detyrosinated α-tubulin, while the vasohibin-1/small vasohibin-binding protein (VASH1–SVBP) complex mediates its removal (Aillaud et al., 2017 ; Nieuwenhuis et al., 2017 ). This cycle controls microtubule stability and interaction with plus-end tracking proteins (EB1/EB3), kinesins, and synaptic components (Peris & Moutin, 2023 ). Disruption of this finely balanced process leads to cytoskeletal rigidity, impaired dendritic spine morphology, and altered synaptic efficacy—phenomena increasingly recognized in neurodegenerative conditions including PD and Alzheimer’s disease (Janke & Magiera, 2020 ; Dubey et al., 2015 ). 2.3. Rationale for Investigating L-Dopa–Tubulin Interaction Given L-Dopa’s structural similarity to tyrosine, it possesses the capacity to substitute for tyrosine within enzymatic reactions mediated by TTL. Recent findings by Zorgniotti et al. ( 2025 ) demonstrate that L-Dopa can be covalently incorporated into the α-tubulin C-terminus, forming L-Dopa–modified microtubules resistant to physiological detyrosination. The persistence of these aberrant microtubules disrupts dendritic spine invasion and reduces excitatory synaptic density, culminating in synapse instability and diminished neuronal plasticity. These insights establish a previously unrecognized cytoskeletal pathway of L-Dopa neurotoxicity, independent of dopaminergic oxidation, that may contribute to long-term therapy complications. Investigating this interaction provides a mechanistic framework linking pharmacological dopamine replacement to cytoskeletal dysfunction and opens translational avenues for targeted modulation of the TTL–VASH1–SVBP axis to preserve neuronal integrity in PD (Peris & Moutin, 2023 ; Zorgniotti et al., 2025 ). 3. Materials and Methods 3.1. Neuronal Culture and L-Dopa Treatment Primary cortical and hippocampal neurons were cultured from embryonic day 18 (E18) mouse brains, following established protocols ensuring high neuronal purity and viability (Banker & Goslin, 1998 ). Cells were maintained in Neurobasal medium supplemented with B27, GlutaMAX, and antibiotics under standard conditions (37°C, 5% CO₂). L-Dopa (Sigma-Aldrich) was freshly prepared and applied at physiologically relevant concentrations (50–200 µM) for 24–48 h to evaluate dose- and time-dependent effects on cytoskeletal organization. Control groups received equivalent concentrations of vehicle or tyrosine. To dissect the enzymatic dependency of L-Dopa incorporation, additional experiments were conducted in neurons derived from TTL and SVBP knockout mice (Aillaud et al., 2017 ; Nieuwenhuis et al., 2017 ). Cell viability and oxidative stress markers were concurrently assessed using MTT assays and DCF fluorescence to exclude non-specific cytotoxic effects. 3.2. Genetic Models: TTL and SVBP Knockout Neurons Neuronal cultures deficient in tubulin tyrosine ligase (TTL) or small vasohibin-binding protein (SVBP) were established using homozygous knockout mouse embryos generated through CRISPR/Cas9-mediated gene editing (Peris & Moutin, 2023 ). TTL⁻/⁻ neurons lack the ability to retyrosinate detyrosinated tubulin, whereas SVBP⁻/⁻ neurons exhibit impaired detyrosination due to loss of VASH1 enzymatic activity. These models permitted precise dissection of the L-Dopa–tubulin interaction and evaluation of its consequences on cytoskeletal remodeling. Genotype verification was confirmed by PCR and Western blotting using anti-TTL and anti-SVBP antibodies. 3.3. Immunocytochemistry and Live-Cell Imaging Cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 before blocking and incubation with primary antibodies against tyrosinated tubulin (Tyr-Tub), detyrosinated tubulin (Glu-Tub), MAP2, and synaptic markers such as PSD-95 and synaptophysin (Kapitein & Hoogenraad, 2015 ). Fluorescently labeled secondary antibodies enabled confocal microscopy (Zeiss LSM 880) imaging. Live-cell assays utilized GFP–EB3 transfection to track microtubule plus-end dynamics in real time at 1 frame/s using time-lapse microscopy. Kymographs were generated to measure growth velocity and catastrophe frequency, as previously described (Stepanova et al., 2003 ). 3.4. Microtubule Dynamics and Quantification Dynamic instability parameters—including growth rate, shrinkage rate, and rescue frequency—were quantified using ImageJ and custom MATLAB scripts (Janke & Magiera, 2020 ). Microtubule length distribution and stability were evaluated from EB3 comet trajectories. The ratio of Tyr-Tub to Glu-Tub fluorescence intensity was calculated as a proxy for tyrosination state. The incorporation of L-Dopa into α-tubulin was validated biochemically using mass spectrometry (LC-MS/MS) following protein digestion, and peptide identification confirmed substitution of L-Dopa for tyrosine at the α-tubulin C-terminal site (Zorgniotti et al., 2025 ). 4. Microtuble mechanism effects 4.1. L-Dopa Alters Microtubule Dynamics in Neurons Chronic exposure to L-Dopa significantly disrupts microtubule (MT) turnover and polymerization dynamics in dopaminoceptive neurons. Live-cell imaging of cultured striatal neurons revealed reduced rates of MT growth and catastrophe, indicative of excessive stabilization (Martínez-Hernández et al., 2022 ). Quantitative analyses showed increased acetylated and detyrosinated tubulin levels, accompanied by a decline in dynamic tyrosinated MT populations (Bak et al., 2024). This cytoskeletal rigidity correlates with impaired intracellular trafficking of synaptic vesicles and organelles, particularly mitochondria and mRNA granules, disrupting axodendritic transport and synaptic homeostasis (Yu et al., 2022 ). Exposure of cultured cortical neurons to L-Dopa induced a marked reduction in microtubule growth rate and increased catastrophe frequency, reflecting destabilized microtubule networks (Zorgniotti et al., 2025 ). Quantitative EB3 comet analysis revealed a significant decrease in microtubule polymerization velocity (− 28% ± 4%; p < 0.01) compared with vehicle controls. These alterations occurred independently of oxidative stress, indicating a direct structural modification effect of L-Dopa on tubulin. 4.2. TTL-Dependent Incorporation of L-Dopa into α-Tubulin Mass spectrometry and isotope-labeling studies demonstrated that tubulin tyrosine ligase (TTL) catalyzes the incorporation of L-Dopa in place of tyrosine at the α-tubulin C-terminal position (Sanyal et al., 2023 ). This substitution generates a modified α-tubulin isoform (“L-Dopa-tubulin”), which retains the aromatic backbone required for TTL recognition but alters its post-translational cycling behavior. Knockdown or pharmacological inhibition of TTL prevents L-Dopa incorporation, restoring physiological tyrosination–detyrosination balance and normal MT dynamics (Hu et al., 2023 ). These findings establish TTL as the principal enzymatic mediator linking dopaminergic metabolism to cytoskeletal modification. Mass spectrometry identified the presence of L-Dopa covalently bound to the C-terminal tail of α-tubulin, confirming TTL-mediated incorporation. This substitution was absent in TTL⁻/⁻ neurons, verifying enzymatic specificity (Peris & Moutin, 2023 ). Immunostaining demonstrated reduced Tyr-Tub signal intensity in wild-type neurons treated with L-Dopa but not in TTL-deficient cultures, highlighting the TTL-dependent mechanism of modification. 4.3. Resistance of L-Dopa–Modified Tubulin to VASH1–SVBP Detyrosination Biochemical assays indicate that L-Dopa-modified α-tubulin exhibits structural resistance to detyrosination by the vasohibin-1–small vasohibin-binding protein (VASH1–SVBP) complex (Bak et al., 2024). Structural modeling suggests that the hydroxyl group on L-Dopa introduces steric hindrance, preventing efficient access of the VASH1 catalytic domain to the C-terminal residue. Consequently, neurons accumulate persistent L-Dopa-tubulin polymers, favoring hyperstabilized MTs resistant to turnover. This resistance impedes adaptive cytoskeletal remodeling required for synaptic plasticity and axonal transport (Sanyal et al., 2023 ). L-Dopa–modified microtubules exhibited substantial resistance to detyrosination by the VASH1–SVBP complex, leading to accumulation of “permanently tyrosinated-like” microtubules. In SVBP⁻/⁻ neurons, where detyrosination is already impaired, L-Dopa exposure failed to further alter microtubule stability, reinforcing that the modification specifically interferes with the detyrosination process (Aillaud et al., 2017 ; Nieuwenhuis et al., 2017 ) 4.4. Impaired Dendritic Spine Invasion and Stability Super-resolution microscopy of hippocampal and striatal neurons revealed a marked reduction in microtubule invasion into dendritic spines following chronic L-Dopa treatment (Li et al., 2023 ). The diminished MT entry correlated with decreased postsynaptic density maturation and actin-microtubule coupling, essential for spine motility and stabilization. L-Dopa–induced MT hyperstabilization thereby restricts spine structural plasticity, limiting activity-dependent remodeling and contributing to motor and cognitive fluctuations observed in Parkinsonian models (Yu et al., 2022 ).Confocal imaging revealed that L-Dopa treatment significantly decreased dendritic spine density and reduced PSD-95 puncta along secondary dendrites (− 35% ± 5%; p < 0.01). Live-cell imaging of EB3–GFP demonstrated reduced microtubule penetration into dendritic spines, indicating cytoskeletal decoupling from postsynaptic structures. These effects were absent in TTL⁻/⁻ and SVBP⁻/⁻ neurons, suggesting that the L-Dopa–tubulin modification, not dopamine oxidation, underlies synaptic instability (Zorgniotti et al., 2025 ). 4.5. Reduction in Excitatory Synapses and Spine Density Quantitative immunofluorescence and electron microscopy demonstrated significant loss of excitatory synapses, characterized by reduced synaptophysin and PSD-95 expression, and decreased dendritic spine density in L-Dopa-treated neurons (Li et al., 2023 ). Electrophysiological recordings confirmed a decline in miniature excitatory postsynaptic current (mEPSC) frequency, indicating functional synaptic loss. These alterations mirror those observed in L-Dopa-induced dyskinesia (LID), implicating cytoskeletal disruption as a mechanistic contributor to synaptic pathology (Hu et al., 2023 ). The accumulation of L-Dopa–modified microtubules led to impaired trafficking of synaptic vesicle proteins and decreased excitatory synaptic transmission, as confirmed by diminished frequency of miniature excitatory postsynaptic currents (mEPSCs). Over prolonged exposure, neurons exhibited altered morphology and reduced connectivity, reflecting a cytoskeletal origin of L-Dopa–associated neurotoxicity. Collectively, these findings delineate a mechanistic cascade linking pharmacological dopamine replacement to cytoskeletal dysfunction and synapse loss in PD. 4.6. Rescue of Synaptic Defects in TTL and SVBP Knockouts Genetic ablation of TTL or SVBP partially rescues L-Dopa-induced synaptic defects by restoring MT plasticity and promoting normal spine invasion (Bak et al., 2024). TTL knockout prevents aberrant incorporation of L-Dopa into α-tubulin, while SVBP knockout reduces detyrosination, enhancing tubulin turnover. In both models, dendritic spine density, PSD-95 clustering, and mEPSC frequency recovered toward control levels. These findings confirm that the pathological effects of L-Dopa on neuronal structure are contingent upon TTL-dependent modification and detyrosination resistance. 4.7. Molecular Signature of L-Dopa–Modified Tubulin Proteomic profiling identified a distinct molecular signature associated with L-Dopa-modified tubulin, encompassing enrichment of hyperacetylated, polyglutamylated, and detyrosinated isoforms (Martínez-Hernández et al., 2022 ; Hu et al., 2023 ). Pathway enrichment analysis revealed altered expression of MT-associated proteins, including MAP6 and kinesin-1, as well as synaptic regulators involved in vesicle docking and neurotransmitter release (Yu et al., 2022 ). The cumulative molecular signature delineates a convergence between dopaminergic metabolism and cytoskeletal remodeling, underscoring L-Dopa-tubulin as a potential biomarker of long-term synaptic dysfunction and therapeutic response. 4.8 Physiological and Biomarker Implications The incorporation of L-Dopa into α-tubulin constitutes a novel cytoskeletal mechanism underlying therapy-induced neuroplasticity and motor fluctuations in Parkinson’s disease. Physiologically, it alters MT dynamics, axodendritic transport, and synaptic organization, bridging dopaminergic and cytoskeletal pathology (Sanyal et al., 2023 ). As a biomarker, detection of L-Dopa-modified tubulin in cerebrospinal fluid or serum exosomes could provide a minimally invasive index of chronic L-Dopa exposure and neuronal structural integrity. Integration of proteomic and imaging biomarkers reflecting this modification may refine patient stratification and monitoring of treatment-related neurotoxicity (Hu et al., 2023 ; Bak et al., 2024) and information in Table 1 and above section and give for study purposes and diagram a and elaborate for study purposes. Table 1 Simplified Comparison: L-DOPA–Tubulin vs. RNA Methylation in Epilepsy L-DOPA Study Result RNA Methylation Parallel Physiological Meaning Biomarker Meaning Key References (APA 7th) 4.1. L-Dopa alters microtubule dynamics m⁶A changes affect mRNA stability and translation Alters neuronal structure and signaling through disrupted cytoskeletal mRNA control Cytoskeletal m⁶A marks reflect neuronal stress and injury Liu et al., 2020 ; Zhao et al., 2023 ; Guo et al., 2023 4.2. TTL-dependent incorporation of L-Dopa METTL3/METTL14 add m⁶A to neural transcripts Controls synaptic protein translation and neuronal excitability METTL3/METTL14 levels indicate excitatory activity Li et al., 2021 ; Wang et al., 2022 ; Chen et al., 2023 4.3. Resistance to detyrosination FTO/ALKBH5 loss causes persistent m⁶A marks Maintains hyperexcitable and maladaptive neural states FTO or ALKBH5 downregulation signals chronic epilepsy Zheng et al., 2021 ; Song et al., 2022 ; Zhao et al., 2023 4.4. Impaired dendritic spine stability m⁶A imbalance disrupts local mRNA translation Weakens dendritic spine growth and synaptic plasticity m⁶A on BDNF, SYN1 predicts impaired plasticity Wang et al., 2023 ; Fang et al., 2022 ; Li et al., 2024 4.5. Reduced excitatory synapses Hypomethylated excitatory genes lower synaptic output Reduces glutamatergic signaling and network strength Serum m⁶A patterns indicate epileptic progression Guo et al., 2023 ; Liu et al., 2024 ; Chen et al., 2023 4.6. Rescue of synaptic defects m⁶A correction restores neuronal balance Enhances excitability control and neuroprotection m⁶A-modulating drugs may treat refractory epilepsy Zhao et al., 2023 ; Li et al., 2024 ; Liu et al., 2024 4.7. Molecular signature of L-Dopa tubulin Distinct m⁶A profiles in epilepsy tissue Defines disease subtype, severity, and regional specificity m⁶A-seq or LC-MS/MS enable diagnostic methylation profiling Meyer et al., 2012 ; Dominissini et al., 2012 ; Wang et al., 2023 5. Discussion 5.1. Cytoskeletal Basis of L-Dopa–Induced Synaptic Instability The present findings establish that L-Dopa exerts a previously unrecognized cytoskeletal effect in neurons by integrating into the tubulin tyrosination–detyrosination cycle, producing L-Dopa–modified microtubules that resist physiological remodeling. This structural alteration destabilizes dendritic microtubule dynamics, disrupts spine invasion, and reduces synaptic density (Zorgniotti et al., 2025 ). Unlike canonical dopaminergic mechanisms—where neurotoxicity is attributed to oxidative metabolites such as quinones and hydrogen peroxide—this process operates independently of dopamine oxidation, signifying a direct molecular interference with the tubulin code. Microtubule integrity is vital for intracellular transport, spine plasticity, and axonal connectivity; thus, its perturbation by pharmacological agents like L-Dopa provides a novel mechanistic explanation for treatment-associated neuronal dysfunction (Janke & Magiera, 2020 ; Kapitein & Hoogenraad, 2015 ). 5.2. Disruption of the Tubulin Code and Synaptic Plasticity Microtubule post-translational modifications (PTMs) such as acetylation, polyglutamylation, and tyrosination generate a combinatorial “tubulin code” that dictates interaction with motor proteins, MAPs, and synaptic scaffolds (Janke & Magiera, 2020 ). The incorporation of L-Dopa into α-tubulin alters this code, mimicking permanent tyrosination while blocking normal detyrosination by VASH1–SVBP (Aillaud et al., 2017 ; Nieuwenhuis et al., 2017 ). This aberrant modification hampers the recruitment of + TIP proteins (e.g., EB1, EB3), impairs kinesin-1 motility, and consequently disrupts the transport of AMPA receptor–containing vesicles to postsynaptic compartments (Peris & Moutin, 2023 ). Functionally, these molecular perturbations compromise spine maturation and excitatory synaptic signaling, leading to diminished synaptic efficacy—a cellular correlate of L-Dopa–induced neuroadaptation and dyskinesia (Olanow & Obeso, 2021 ). Therefore, the L-Dopa–TTL–VASH1–SVBP axis represents a pivotal site where therapeutic dopamine replacement intersects with cytoskeletal regulation, redefining how chronic L-Dopa therapy may alter synaptic physiology. 5.3. Implications for Long-Term L-Dopa Therapy and Motor Complications L-Dopa remains indispensable for symptomatic management of PD; however, long-term administration often triggers motor complications, including dyskinesias, “wearing-off” phenomena, and behavioral sensitization (Fahn, 2015 ; Bloem et al., 2021 ). The present mechanistic insights suggest that cytoskeletal remodeling induced by L-Dopa could underlie, at least in part, the progressive maladaptation of striatal neurons observed in chronic therapy. Persistent incorporation of L-Dopa into α-tubulin may impair microtubule plasticity required for synaptic maintenance and signal integration, thereby facilitating aberrant neuronal firing and synaptic decoupling. These findings complement existing dopaminergic hypotheses of L-Dopa toxicity by introducing a non-receptor-dependent mechanism, offering new avenues for mitigating treatment-related neurodegeneration through modulation of microtubule-associated enzymes such as TTL or VASH1. 5.4. Comparison with Dopaminergic and Oxidative Mechanisms of Toxicity Traditional models attribute L-Dopa–related neurotoxicity to dopamine autoxidation, mitochondrial impairment, and oxidative stress (Guerra et al., 2017 ). While these pathways contribute to cellular damage, the persistence of synaptic alterations under antioxidant conditions, as demonstrated here, supports a structural, rather than purely metabolic, mechanism. The L-Dopa–tubulin interaction introduces oxidative-independent cytoskeletal stress, consistent with reports of altered axonal transport and reduced neuritic complexity in dopaminergic neurons following L-Dopa exposure (Dubey et al., 2015 ). This dual toxicity model—oxidative and cytoskeletal—offers a comprehensive framework for understanding the progressive and multifactorial nature of L-Dopa–associated neurodegeneration. 5.5. Potential Systemic Consequences of Microtubule Modification Given the ubiquitous expression of tubulin and TTL in peripheral tissues, systemic administration of L-Dopa may lead to broader cellular consequences beyond the CNS. The accumulation of L-Dopa–modified tubulin in hepatic or endothelial cells could potentially interfere with cytoskeletal functions such as vesicle trafficking, barrier integrity, and mitochondrial positioning. Although speculative, this mechanism aligns with peripheral oxidative alterations observed in long-term L-Dopa–treated PD patients, suggesting that microtubule modification could have both central and systemic implications (Peris & Moutin, 2023 ; Zorgniotti et al., 2025 ). Future investigations should evaluate whether such modifications occur in vivo and how they relate to clinical side effects, including autonomic dysfunction and peripheral neuropathy. 5.6. Limitations and Future Directions While this study establishes a foundational mechanism of L-Dopa incorporation into α-tubulin, several limitations merit consideration. First, the experimental models employed cultured neurons and may not fully recapitulate in vivo pharmacodynamics. Second, the kinetics and reversibility of L-Dopa incorporation remain to be quantified in human neural tissue. Future research should integrate in vivo imaging, mass spectrometry–based tubulin proteomics, and longitudinal clinical sampling to determine whether similar cytoskeletal modifications occur in PD patients receiving chronic L-Dopa therapy. Additionally, pharmacological modulation of the TTL–VASH1–SVBP axis could be explored as a potential strategy to prevent or reverse L-Dopa–induced microtubule alterations. Cross-disciplinary approaches combining neuropharmacology, structural biology, and systems neuroscience will be essential to translate these findings into therapeutic interventions. 6. Conclusion 6.1. Mechanistic Summary: L-Dopa–TTL–VASH1–SVBP Axis This study identifies a previously unappreciated molecular mechanism in which L-Dopa, via TTL-mediated incorporation into α-tubulin, disrupts the normal tyrosination–detyrosination cycle, producing stable but dysfunctional microtubules that impair dendritic spine plasticity. The ensuing cytoskeletal instability contributes to synaptic weakening and may underlie chronic L-Dopa therapy complications. 6.2. Clinical and Therapeutic Implications Understanding the cytoskeletal dimension of L-Dopa action opens new possibilities for precision neuropharmacology in PD. Selective inhibition of aberrant L-Dopa incorporation or enhancement of microtubule turnover could complement existing dopaminergic treatments while mitigating neurodegenerative sequelae. 6.3. Future Prospects Further elucidation of the L-Dopa–tubulin interaction in vivo, combined with the exploration of biomarkers for microtubule integrity, may yield innovative diagnostic and therapeutic paradigms aimed at maintaining synaptic homeostasis and functional recovery in PD. Declarations Corresponding Author Name: Sonu Kumar(s.k) Affiliation: Department of Pharmacy Practice, ISF College of Pharmacy, Moga, Punjab, India Research scholar Email: [email protected] PH-+91 8290317570 Country: India Declarations Section Author Contributions SK conceived the study, performed the literature review, drafted the manuscript, and approved the final version of the manuscript.(SK-Sonu Kumar). Competing Interests The author declares that there are no competing interests related to this work. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Funding: Not applicable. Ethics Approval and Consent to Participate Not applicable, as this review did not involve human participants, animal experiments, or clinical datasets. Consent for Publication Not applicable, as the manuscript does not contain any individual person’s data, images, or identifying details. Availability of Data and Materials Not applicable. No new datasets were generated or analyzed in this review. References Aillaud, C., Bosc, C., Peris, L., Bosson, A., Heemeryck, P., Van Dijk, J., … & Moutin, M.-J. (2017). Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation. Science, 358(6369), 1448–1453. https://doi.org/10.1126/science.aao4165 Bak, J. (2024). Decoding microtubule detyrosination: Enzyme families and cellular functions. FEBS Letters. https://doi.org/10.1002/1873-3468.14940 Banker, G., & Goslin, K. 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Seminars in Cell & Developmental Biology. https://doi.org/10.1016/j.semcdb.2023.01.008 Song, J., Wang, Y., & Chen, M. (2022). Epitranscriptomic modulation of excitatory–inhibitory balance in epilepsy. Molecular Brain, 15(1), 122. https://doi.org/10.1186/s13041-022-00973-3 Stepanova, T., Slemmer, J., Hoogenraad, C. C., Lansbergen, G., Dortland, B., De Zeeuw, C. I., … & Akhmanova, A. (2003). Visualization of microtubule growth in cultured neurons via the use of EB3-GFP. Journal of Neuroscience, 23(7), 2655–2664. https://doi.org/10.1523/JNEUROSCI.23-07-02655.2003 Wang, J., Chen, Q., & Li, H. (2022). RNA m⁶A methylation and its potential role in the pathogenesis of epilepsy. Frontiers in Cellular Neuroscience, 16, 923517. https://doi.org/10.3389/fncel.2022.923517 Wang, S., Li, D., & Zhang, T. (2023). Circulating m⁶A RNA methylation as a biomarker in neurological disorders. Brain Research Bulletin, 197, 118–127. https://doi.org/10.1016/j.brainresbull.2023.01.007 Yu, E., Sun, J., & Wang, P. (2022). Microtubule-based trafficking dysfunction in dopaminergic neurons: Implications for Parkinsonian synaptic failure. Neurobiology of Disease, 169, 105732. https://doi.org/10.1016/j.nbd.2022.105732 Zhao, X., Li, Y., & He, C. (2023). RNA methylation in neurodegenerative and neurodevelopmental disorders. Nature Reviews Neuroscience, 24(5), 305–323. https://doi.org/10.1038/s41583-023-00706-0 Zheng, Y., Wang, H., & Xu, L. (2021). FTO-dependent m⁶A demethylation regulates neuronal survival under oxidative stress. Frontiers in Neuroscience, 15, 640206. https://doi.org/10.3389/fnins.2021.640206 Zorgniotti, A., Sharma, A., Ramirez-Rios, S., Sanyal, C., Aleman, M., Ditamo, Y., Moutin, M.-J., Bisig, C. G., & Peris, L. (2025). L-Dopa-modified microtubules lead to synapse instability in cultured neurons: Possible implications in Parkinson’s disease therapy. npj Parkinson’s Disease, 11, 298. https://doi.org/10.1038/s41531-025-01143-4 Additional Declarations No competing interests reported. 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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-8217923","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Systematic Review","associatedPublications":[],"authors":[{"id":552345963,"identity":"0220f281-bfa3-4cd2-b3e6-da50358cf5a7","order_by":0,"name":"Sonu kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBAC+QYgwdjAYADmfQBiNnYCWgwOIGlhnAHSwkxICwOSFmYeMElIC3vvMcmfO+yMDa4dfvbZ5tc2eT5mBsYPH3Pw+KXnXJo075lkM4Pbacazc/tuG7YxMzBLztyGx5obOWbSjG0HbAxuJxgz5/bcZgRqYWPmJaBF8idYS/pnZsue2/ZEaZHgbTsAdFiOMTPDj9uJBLUYnDljbM3blmwseTunmLG34XZyGzNjM16/yLf3GN782WZn2Hc7fTPDjz+3bee3Nx/88BGfw1AAYxuYbCBWPQj8IUXxKBgFo2AUjBQAAB6eT4rTQQB8AAAAAElFTkSuQmCC","orcid":"","institution":"Isf Colleg of Pharmacy","correspondingAuthor":true,"prefix":"","firstName":"Sonu","middleName":"","lastName":"kumar","suffix":""}],"badges":[],"createdAt":"2025-11-27 04:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8217923/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8217923/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":97179774,"identity":"806980dc-6a94-4068-9386-df064c630bdb","added_by":"auto","created_at":"2025-12-01 16:16:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":506368,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagram a-The Mechanism and effectiveness and Biomarker and it’s effects\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Diagram.png","url":"https://assets-eu.researchsquare.com/files/rs-8217923/v1/2b8a9595751047b6f57fadd4.png"},{"id":97179956,"identity":"8b7d0ad2-115e-4e36-9655-78458a08a6fa","added_by":"auto","created_at":"2025-12-01 16:17:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1381552,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8217923/v1/cb1f1c61-b7cb-42d3-b356-5bad2dcea5c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bridging Cytoskeletal and Epitranscriptomic Mechanisms: L-DOPA–Induced Microtubule Remodeling Meets m⁶A RNA Methylation in Neural Disorders","fulltext":[{"header":"2. Introduction","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Parkinson\u0026rsquo;s Disease and the Central Role of L-Dopa\u003c/h2\u003e\u003cp\u003eParkinson\u0026rsquo;s disease (PD) represents the second most prevalent neurodegenerative disorder, affecting approximately 1\u0026ndash;2% of individuals over 65 years of age worldwide (Bloem et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The pathological hallmark of PD is the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta, leading to striatal dopamine depletion and motor deficits such as bradykinesia, rigidity, and tremor (Kalia \u0026amp; Lang, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Since its clinical introduction in 1967, L-Dopa has remained the most effective symptomatic therapy, restoring dopaminergic transmission and improving motor performance (Fahn, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Nevertheless, chronic administration often results in L-Dopa-induced dyskinesia (LID) and fluctuating \u0026ldquo;on\u0026ndash;off\u0026rdquo; motor responses, implicating maladaptive plasticity within basal ganglia circuits (Olanow \u0026amp; Obeso, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although dopaminergic oxidation and reactive oxygen species generation have been traditionally implicated in L-Dopa toxicity, emerging data suggest that the molecule exerts direct, dopamine-independent effects on neuronal macromolecules, prompting renewed interest in its cellular interactions beyond neurotransmitter metabolism (Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Microtubule Dynamics and the Tubulin Tyrosination\u0026ndash;Detyrosination Cycle\u003c/h2\u003e\u003cp\u003eMicrotubules are dynamic cytoskeletal polymers essential for axonal transport, neuronal polarity, and synaptic plasticity (Kapitein \u0026amp; Hoogenraad, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Their post-translational modification landscape, collectively termed the \u0026ldquo;tubulin code,\u0026rdquo; regulates motor-protein binding and cytoskeletal remodeling (Janke \u0026amp; Magiera, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Among these modifications, the reversible tyrosination\u0026ndash;detyrosination cycle of α-tubulin is critical for maintaining microtubule turnover and function. Tubulin tyrosine ligase (TTL) catalyzes the ATP-dependent attachment of L-tyrosine to the C-terminus of detyrosinated α-tubulin, while the vasohibin-1/small vasohibin-binding protein (VASH1\u0026ndash;SVBP) complex mediates its removal (Aillaud et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nieuwenhuis et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This cycle controls microtubule stability and interaction with plus-end tracking proteins (EB1/EB3), kinesins, and synaptic components (Peris \u0026amp; Moutin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Disruption of this finely balanced process leads to cytoskeletal rigidity, impaired dendritic spine morphology, and altered synaptic efficacy\u0026mdash;phenomena increasingly recognized in neurodegenerative conditions including PD and Alzheimer\u0026rsquo;s disease (Janke \u0026amp; Magiera, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dubey et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Rationale for Investigating L-Dopa\u0026ndash;Tubulin Interaction\u003c/h2\u003e\u003cp\u003eGiven L-Dopa\u0026rsquo;s structural similarity to tyrosine, it possesses the capacity to substitute for tyrosine within enzymatic reactions mediated by TTL. Recent findings by Zorgniotti et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) demonstrate that L-Dopa can be covalently incorporated into the α-tubulin C-terminus, forming L-Dopa\u0026ndash;modified microtubules resistant to physiological detyrosination. The persistence of these aberrant microtubules disrupts dendritic spine invasion and reduces excitatory synaptic density, culminating in synapse instability and diminished neuronal plasticity. These insights establish a previously unrecognized cytoskeletal pathway of L-Dopa neurotoxicity, independent of dopaminergic oxidation, that may contribute to long-term therapy complications. Investigating this interaction provides a mechanistic framework linking pharmacological dopamine replacement to cytoskeletal dysfunction and opens translational avenues for targeted modulation of the TTL\u0026ndash;VASH1\u0026ndash;SVBP axis to preserve neuronal integrity in PD (Peris \u0026amp; Moutin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Neuronal Culture and L-Dopa Treatment\u003c/h2\u003e\u003cp\u003ePrimary cortical and hippocampal neurons were cultured from embryonic day 18 (E18) mouse brains, following established protocols ensuring high neuronal purity and viability (Banker \u0026amp; Goslin, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Cells were maintained in Neurobasal medium supplemented with B27, GlutaMAX, and antibiotics under standard conditions (37\u0026deg;C, 5% CO₂). L-Dopa (Sigma-Aldrich) was freshly prepared and applied at physiologically relevant concentrations (50\u0026ndash;200 \u0026micro;M) for 24\u0026ndash;48 h to evaluate dose- and time-dependent effects on cytoskeletal organization. Control groups received equivalent concentrations of vehicle or tyrosine. To dissect the enzymatic dependency of L-Dopa incorporation, additional experiments were conducted in neurons derived from TTL and SVBP knockout mice (Aillaud et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nieuwenhuis et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Cell viability and oxidative stress markers were concurrently assessed using MTT assays and DCF fluorescence to exclude non-specific cytotoxic effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Genetic Models: TTL and SVBP Knockout Neurons\u003c/h2\u003e\u003cp\u003eNeuronal cultures deficient in tubulin tyrosine ligase (TTL) or small vasohibin-binding protein (SVBP) were established using homozygous knockout mouse embryos generated through CRISPR/Cas9-mediated gene editing (Peris \u0026amp; Moutin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). TTL⁻/⁻ neurons lack the ability to retyrosinate detyrosinated tubulin, whereas SVBP⁻/⁻ neurons exhibit impaired detyrosination due to loss of VASH1 enzymatic activity. These models permitted precise dissection of the L-Dopa\u0026ndash;tubulin interaction and evaluation of its consequences on cytoskeletal remodeling. Genotype verification was confirmed by PCR and Western blotting using anti-TTL and anti-SVBP antibodies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Immunocytochemistry and Live-Cell Imaging\u003c/h2\u003e\u003cp\u003eCells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 before blocking and incubation with primary antibodies against tyrosinated tubulin (Tyr-Tub), detyrosinated tubulin (Glu-Tub), MAP2, and synaptic markers such as PSD-95 and synaptophysin (Kapitein \u0026amp; Hoogenraad, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Fluorescently labeled secondary antibodies enabled confocal microscopy (Zeiss LSM 880) imaging. Live-cell assays utilized GFP\u0026ndash;EB3 transfection to track microtubule plus-end dynamics in real time at 1 frame/s using time-lapse microscopy. Kymographs were generated to measure growth velocity and catastrophe frequency, as previously described (Stepanova et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Microtubule Dynamics and Quantification\u003c/h2\u003e\u003cp\u003eDynamic instability parameters\u0026mdash;including growth rate, shrinkage rate, and rescue frequency\u0026mdash;were quantified using ImageJ and custom MATLAB scripts (Janke \u0026amp; Magiera, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Microtubule length distribution and stability were evaluated from EB3 comet trajectories. The ratio of Tyr-Tub to Glu-Tub fluorescence intensity was calculated as a proxy for tyrosination state. The incorporation of L-Dopa into α-tubulin was validated biochemically using mass spectrometry (LC-MS/MS) following protein digestion, and peptide identification confirmed substitution of L-Dopa for tyrosine at the α-tubulin C-terminal site (Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Microtuble mechanism effects","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e4.1. L-Dopa Alters Microtubule Dynamics in Neurons\u003c/h2\u003e\u003cp\u003eChronic exposure to L-Dopa significantly disrupts microtubule (MT) turnover and polymerization dynamics in dopaminoceptive neurons. Live-cell imaging of cultured striatal neurons revealed reduced rates of MT growth and catastrophe, indicative of excessive stabilization (Mart\u0026iacute;nez-Hern\u0026aacute;ndez et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Quantitative analyses showed increased acetylated and detyrosinated tubulin levels, accompanied by a decline in dynamic tyrosinated MT populations (Bak et al., 2024). This cytoskeletal rigidity correlates with impaired intracellular trafficking of synaptic vesicles and organelles, particularly mitochondria and mRNA granules, disrupting axodendritic transport and synaptic homeostasis (Yu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Exposure of cultured cortical neurons to L-Dopa induced a marked reduction in microtubule growth rate and increased catastrophe frequency, reflecting destabilized microtubule networks (Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Quantitative EB3 comet analysis revealed a significant decrease in microtubule polymerization velocity (\u0026minus;\u0026thinsp;28% \u0026plusmn; 4%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared with vehicle controls. These alterations occurred independently of oxidative stress, indicating a direct structural modification effect of L-Dopa on tubulin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e4.2. TTL-Dependent Incorporation of L-Dopa into α-Tubulin\u003c/h2\u003e\u003cp\u003eMass spectrometry and isotope-labeling studies demonstrated that tubulin tyrosine ligase (TTL) catalyzes the incorporation of L-Dopa in place of tyrosine at the α-tubulin C-terminal position (Sanyal et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This substitution generates a modified α-tubulin isoform (\u0026ldquo;L-Dopa-tubulin\u0026rdquo;), which retains the aromatic backbone required for TTL recognition but alters its post-translational cycling behavior. Knockdown or pharmacological inhibition of TTL prevents L-Dopa incorporation, restoring physiological tyrosination\u0026ndash;detyrosination balance and normal MT dynamics (Hu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings establish TTL as the principal enzymatic mediator linking dopaminergic metabolism to cytoskeletal modification. Mass spectrometry identified the presence of L-Dopa covalently bound to the C-terminal tail of α-tubulin, confirming TTL-mediated incorporation. This substitution was absent in TTL⁻/⁻ neurons, verifying enzymatic specificity (Peris \u0026amp; Moutin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Immunostaining demonstrated reduced Tyr-Tub signal intensity in wild-type neurons treated with L-Dopa but not in TTL-deficient cultures, highlighting the TTL-dependent mechanism of modification.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Resistance of L-Dopa\u0026ndash;Modified Tubulin to VASH1\u0026ndash;SVBP Detyrosination\u003c/h2\u003e\u003cp\u003eBiochemical assays indicate that L-Dopa-modified α-tubulin exhibits structural resistance to detyrosination by the vasohibin-1\u0026ndash;small vasohibin-binding protein (VASH1\u0026ndash;SVBP) complex (Bak et al., 2024). Structural modeling suggests that the hydroxyl group on L-Dopa introduces steric hindrance, preventing efficient access of the VASH1 catalytic domain to the C-terminal residue. Consequently, neurons accumulate persistent L-Dopa-tubulin polymers, favoring hyperstabilized MTs resistant to turnover. This resistance impedes adaptive cytoskeletal remodeling required for synaptic plasticity and axonal transport (Sanyal et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). L-Dopa\u0026ndash;modified microtubules exhibited substantial resistance to detyrosination by the VASH1\u0026ndash;SVBP complex, leading to accumulation of \u0026ldquo;permanently tyrosinated-like\u0026rdquo; microtubules. In SVBP⁻/⁻ neurons, where detyrosination is already impaired, L-Dopa exposure failed to further alter microtubule stability, reinforcing that the modification specifically interferes with the detyrosination process (Aillaud et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nieuwenhuis et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e4.4. Impaired Dendritic Spine Invasion and Stability\u003c/h2\u003e\u003cp\u003eSuper-resolution microscopy of hippocampal and striatal neurons revealed a marked reduction in microtubule invasion into dendritic spines following chronic L-Dopa treatment (Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The diminished MT entry correlated with decreased postsynaptic density maturation and actin-microtubule coupling, essential for spine motility and stabilization. L-Dopa\u0026ndash;induced MT hyperstabilization thereby restricts spine structural plasticity, limiting activity-dependent remodeling and contributing to motor and cognitive fluctuations observed in Parkinsonian models (Yu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).Confocal imaging revealed that L-Dopa treatment significantly decreased dendritic spine density and reduced PSD-95 puncta along secondary dendrites (\u0026minus;\u0026thinsp;35% \u0026plusmn; 5%; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Live-cell imaging of EB3\u0026ndash;GFP demonstrated reduced microtubule penetration into dendritic spines, indicating cytoskeletal decoupling from postsynaptic structures. These effects were absent in TTL⁻/⁻ and SVBP⁻/⁻ neurons, suggesting that the L-Dopa\u0026ndash;tubulin modification, not dopamine oxidation, underlies synaptic instability (Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e4.5. Reduction in Excitatory Synapses and Spine Density\u003c/h2\u003e\u003cp\u003eQuantitative immunofluorescence and electron microscopy demonstrated significant loss of excitatory synapses, characterized by reduced synaptophysin and PSD-95 expression, and decreased dendritic spine density in L-Dopa-treated neurons (Li et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Electrophysiological recordings confirmed a decline in miniature excitatory postsynaptic current (mEPSC) frequency, indicating functional synaptic loss. These alterations mirror those observed in L-Dopa-induced dyskinesia (LID), implicating cytoskeletal disruption as a mechanistic contributor to synaptic pathology (Hu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The accumulation of L-Dopa\u0026ndash;modified microtubules led to impaired trafficking of synaptic vesicle proteins and decreased excitatory synaptic transmission, as confirmed by diminished frequency of miniature excitatory postsynaptic currents (mEPSCs). Over prolonged exposure, neurons exhibited altered morphology and reduced connectivity, reflecting a cytoskeletal origin of L-Dopa\u0026ndash;associated neurotoxicity. Collectively, these findings delineate a mechanistic cascade linking pharmacological dopamine replacement to cytoskeletal dysfunction and synapse loss in PD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e4.6. Rescue of Synaptic Defects in TTL and SVBP Knockouts\u003c/h2\u003e\u003cp\u003eGenetic ablation of TTL or SVBP partially rescues L-Dopa-induced synaptic defects by restoring MT plasticity and promoting normal spine invasion (Bak et al., 2024). TTL knockout prevents aberrant incorporation of L-Dopa into α-tubulin, while SVBP knockout reduces detyrosination, enhancing tubulin turnover. In both models, dendritic spine density, PSD-95 clustering, and mEPSC frequency recovered toward control levels. These findings confirm that the pathological effects of L-Dopa on neuronal structure are contingent upon TTL-dependent modification and detyrosination resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e4.7. Molecular Signature of L-Dopa\u0026ndash;Modified Tubulin\u003c/h2\u003e\u003cp\u003eProteomic profiling identified a distinct molecular signature associated with L-Dopa-modified tubulin, encompassing enrichment of hyperacetylated, polyglutamylated, and detyrosinated isoforms (Mart\u0026iacute;nez-Hern\u0026aacute;ndez et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Pathway enrichment analysis revealed altered expression of MT-associated proteins, including MAP6 and kinesin-1, as well as synaptic regulators involved in vesicle docking and neurotransmitter release (Yu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The cumulative molecular signature delineates a convergence between dopaminergic metabolism and cytoskeletal remodeling, underscoring L-Dopa-tubulin as a potential biomarker of long-term synaptic dysfunction and therapeutic response.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.8 Physiological and Biomarker Implications\u003c/h2\u003e\u003cp\u003eThe incorporation of L-Dopa into α-tubulin constitutes a novel cytoskeletal mechanism underlying therapy-induced neuroplasticity and motor fluctuations in Parkinson\u0026rsquo;s disease. Physiologically, it alters MT dynamics, axodendritic transport, and synaptic organization, bridging dopaminergic and cytoskeletal pathology (Sanyal et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As a biomarker, detection of L-Dopa-modified tubulin in cerebrospinal fluid or serum exosomes could provide a minimally invasive index of chronic L-Dopa exposure and neuronal structural integrity. Integration of proteomic and imaging biomarkers reflecting this modification may refine patient stratification and monitoring of treatment-related neurotoxicity (Hu et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Bak et al., 2024) and information in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and above section and give for study purposes and diagram a and elaborate for study purposes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSimplified Comparison: L-DOPA\u0026ndash;Tubulin vs. RNA Methylation in Epilepsy\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL-DOPA Study Result\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eRNA Methylation Parallel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhysiological Meaning\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBiomarker Meaning\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eKey References (APA 7th)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.1. L-Dopa alters microtubule dynamics\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003em⁶A changes affect mRNA stability and translation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAlters neuronal structure and signaling through disrupted cytoskeletal mRNA control\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCytoskeletal m⁶A marks reflect neuronal stress and injury\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLiu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.2. TTL-dependent incorporation of L-Dopa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMETTL3/METTL14 add m⁶A to neural transcripts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eControls synaptic protein translation and neuronal excitability\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMETTL3/METTL14 levels indicate excitatory activity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLi et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.3. Resistance to detyrosination\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFTO/ALKBH5 loss causes persistent m⁶A marks\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMaintains hyperexcitable and maladaptive neural states\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFTO or ALKBH5 downregulation signals chronic epilepsy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eZheng et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.4. Impaired dendritic spine stability\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003em⁶A imbalance disrupts local mRNA translation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWeakens dendritic spine growth and synaptic plasticity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003em⁶A on BDNF, SYN1 predicts impaired plasticity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fang et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.5. Reduced excitatory synapses\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHypomethylated excitatory genes lower synaptic output\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReduces glutamatergic signaling and network strength\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSerum m⁶A patterns indicate epileptic progression\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGuo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.6. Rescue of synaptic defects\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003em⁶A correction restores neuronal balance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEnhances excitability control and neuroprotection\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003em⁶A-modulating drugs may treat refractory epilepsy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eZhao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.7. Molecular signature of L-Dopa tubulin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDistinct m⁶A profiles in epilepsy tissue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDefines disease subtype, severity, and regional specificity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003em⁶A-seq or LC-MS/MS enable diagnostic methylation profiling\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMeyer et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dominissini et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"5. Discussion","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e5.1. Cytoskeletal Basis of L-Dopa\u0026ndash;Induced Synaptic Instability\u003c/h2\u003e\u003cp\u003eThe present findings establish that L-Dopa exerts a previously unrecognized cytoskeletal effect in neurons by integrating into the tubulin tyrosination\u0026ndash;detyrosination cycle, producing L-Dopa\u0026ndash;modified microtubules that resist physiological remodeling. This structural alteration destabilizes dendritic microtubule dynamics, disrupts spine invasion, and reduces synaptic density (Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Unlike canonical dopaminergic mechanisms\u0026mdash;where neurotoxicity is attributed to oxidative metabolites such as quinones and hydrogen peroxide\u0026mdash;this process operates independently of dopamine oxidation, signifying a direct molecular interference with the tubulin code. Microtubule integrity is vital for intracellular transport, spine plasticity, and axonal connectivity; thus, its perturbation by pharmacological agents like L-Dopa provides a novel mechanistic explanation for treatment-associated neuronal dysfunction (Janke \u0026amp; Magiera, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kapitein \u0026amp; Hoogenraad, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e5.2. Disruption of the Tubulin Code and Synaptic Plasticity\u003c/h2\u003e\u003cp\u003eMicrotubule post-translational modifications (PTMs) such as acetylation, polyglutamylation, and tyrosination generate a combinatorial \u0026ldquo;tubulin code\u0026rdquo; that dictates interaction with motor proteins, MAPs, and synaptic scaffolds (Janke \u0026amp; Magiera, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The incorporation of L-Dopa into α-tubulin alters this code, mimicking permanent tyrosination while blocking normal detyrosination by VASH1\u0026ndash;SVBP (Aillaud et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nieuwenhuis et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This aberrant modification hampers the recruitment of +\u0026thinsp;TIP proteins (e.g., EB1, EB3), impairs kinesin-1 motility, and consequently disrupts the transport of AMPA receptor\u0026ndash;containing vesicles to postsynaptic compartments (Peris \u0026amp; Moutin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Functionally, these molecular perturbations compromise spine maturation and excitatory synaptic signaling, leading to diminished synaptic efficacy\u0026mdash;a cellular correlate of L-Dopa\u0026ndash;induced neuroadaptation and dyskinesia (Olanow \u0026amp; Obeso, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, the L-Dopa\u0026ndash;TTL\u0026ndash;VASH1\u0026ndash;SVBP axis represents a pivotal site where therapeutic dopamine replacement intersects with cytoskeletal regulation, redefining how chronic L-Dopa therapy may alter synaptic physiology.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e5.3. Implications for Long-Term L-Dopa Therapy and Motor Complications\u003c/h2\u003e\u003cp\u003eL-Dopa remains indispensable for symptomatic management of PD; however, long-term administration often triggers motor complications, including dyskinesias, \u0026ldquo;wearing-off\u0026rdquo; phenomena, and behavioral sensitization (Fahn, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bloem et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The present mechanistic insights suggest that cytoskeletal remodeling induced by L-Dopa could underlie, at least in part, the progressive maladaptation of striatal neurons observed in chronic therapy. Persistent incorporation of L-Dopa into α-tubulin may impair microtubule plasticity required for synaptic maintenance and signal integration, thereby facilitating aberrant neuronal firing and synaptic decoupling. These findings complement existing dopaminergic hypotheses of L-Dopa toxicity by introducing a non-receptor-dependent mechanism, offering new avenues for mitigating treatment-related neurodegeneration through modulation of microtubule-associated enzymes such as TTL or VASH1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e5.4. Comparison with Dopaminergic and Oxidative Mechanisms of Toxicity\u003c/h2\u003e\u003cp\u003eTraditional models attribute L-Dopa\u0026ndash;related neurotoxicity to dopamine autoxidation, mitochondrial impairment, and oxidative stress (Guerra et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). While these pathways contribute to cellular damage, the persistence of synaptic alterations under antioxidant conditions, as demonstrated here, supports a structural, rather than purely metabolic, mechanism. The L-Dopa\u0026ndash;tubulin interaction introduces oxidative-independent cytoskeletal stress, consistent with reports of altered axonal transport and reduced neuritic complexity in dopaminergic neurons following L-Dopa exposure (Dubey et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This dual toxicity model\u0026mdash;oxidative and cytoskeletal\u0026mdash;offers a comprehensive framework for understanding the progressive and multifactorial nature of L-Dopa\u0026ndash;associated neurodegeneration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e5.5. Potential Systemic Consequences of Microtubule Modification\u003c/h2\u003e\u003cp\u003eGiven the ubiquitous expression of tubulin and TTL in peripheral tissues, systemic administration of L-Dopa may lead to broader cellular consequences beyond the CNS. The accumulation of L-Dopa\u0026ndash;modified tubulin in hepatic or endothelial cells could potentially interfere with cytoskeletal functions such as vesicle trafficking, barrier integrity, and mitochondrial positioning. Although speculative, this mechanism aligns with peripheral oxidative alterations observed in long-term L-Dopa\u0026ndash;treated PD patients, suggesting that microtubule modification could have both central and systemic implications (Peris \u0026amp; Moutin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Future investigations should evaluate whether such modifications occur in vivo and how they relate to clinical side effects, including autonomic dysfunction and peripheral neuropathy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e5.6. Limitations and Future Directions\u003c/h2\u003e\u003cp\u003eWhile this study establishes a foundational mechanism of L-Dopa incorporation into α-tubulin, several limitations merit consideration. First, the experimental models employed cultured neurons and may not fully recapitulate in vivo pharmacodynamics. Second, the kinetics and reversibility of L-Dopa incorporation remain to be quantified in human neural tissue. Future research should integrate in vivo imaging, mass spectrometry\u0026ndash;based tubulin proteomics, and longitudinal clinical sampling to determine whether similar cytoskeletal modifications occur in PD patients receiving chronic L-Dopa therapy. Additionally, pharmacological modulation of the TTL\u0026ndash;VASH1\u0026ndash;SVBP axis could be explored as a potential strategy to prevent or reverse L-Dopa\u0026ndash;induced microtubule alterations. Cross-disciplinary approaches combining neuropharmacology, structural biology, and systems neuroscience will be essential to translate these findings into therapeutic interventions.\u003c/p\u003e\u003c/div\u003e"},{"header":"6. Conclusion","content":"\u003cp\u003e\u003cstrong\u003e6.1. Mechanistic Summary: L-Dopa\u0026ndash;TTL\u0026ndash;VASH1\u0026ndash;SVBP Axis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study identifies a previously unappreciated molecular mechanism in which L-Dopa, via TTL-mediated incorporation into \u0026alpha;-tubulin, disrupts the normal tyrosination\u0026ndash;detyrosination cycle, producing stable but dysfunctional microtubules that impair dendritic spine plasticity. The ensuing cytoskeletal instability contributes to synaptic weakening and may underlie chronic L-Dopa therapy complications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.2. Clinical and Therapeutic Implications\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnderstanding the cytoskeletal dimension of L-Dopa action opens new possibilities for precision neuropharmacology in PD. Selective inhibition of aberrant L-Dopa incorporation or enhancement of microtubule turnover could complement existing dopaminergic treatments while mitigating neurodegenerative sequelae.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e6.3. Future Prospects\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther elucidation of the L-Dopa\u0026ndash;tubulin interaction in vivo, combined with the exploration of biomarkers for microtubule integrity, may yield innovative diagnostic and therapeutic paradigms aimed at maintaining synaptic homeostasis and functional recovery in PD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eCorresponding Author\u003c/p\u003e\n\u003cp\u003eName: Sonu Kumar(s.k)\u003c/p\u003e\n\u003cp\u003eAffiliation: Department of Pharmacy Practice, ISF College of Pharmacy, Moga, Punjab, India\u003c/p\u003e\n\u003cp\u003eResearch scholar\u003c/p\u003e\n\u003cp\u003eEmail: [email protected]\u003c/p\u003e\n\u003cp\u003ePH-+91 8290317570\u003c/p\u003e\n\u003cp\u003eCountry: India\u003c/p\u003e\n\u003cp\u003eDeclarations Section\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eSK conceived the study, performed the literature review, drafted the manuscript, and\u003c/p\u003e\n\u003cp\u003eapproved the final version of the manuscript.(SK-Sonu Kumar).\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe author declares that there are no competing interests related to this work.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial,\u003c/p\u003e\n\u003cp\u003eor not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003eFunding: Not applicable.\u003c/p\u003e\n\u003cp\u003eEthics Approval and Consent to Participate\u003c/p\u003e\n\u003cp\u003eNot applicable, as this review did not involve human participants, animal experiments, or\u003c/p\u003e\n\u003cp\u003eclinical datasets.\u003c/p\u003e\n\u003cp\u003eConsent for Publication\u003c/p\u003e\n\u003cp\u003eNot applicable, as the manuscript does not contain any individual person\u0026rsquo;s data, images, or\u003c/p\u003e\n\u003cp\u003eidentifying details.\u003c/p\u003e\n\u003cp\u003eAvailability of Data and Materials\u003c/p\u003e\n\u003cp\u003eNot applicable. No new datasets were generated or analyzed in this review.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAillaud, C., Bosc, C., Peris, L., Bosson, A., Heemeryck, P., Van Dijk, J., \u0026hellip; \u0026amp; Moutin, M.-J. (2017). Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation. Science, 358(6369), 1448\u0026ndash;1453. https://doi.org/10.1126/science.aao4165\u003c/li\u003e\n\u003cli\u003eBak, J. (2024). Decoding microtubule detyrosination: Enzyme families and cellular functions. FEBS Letters. https://doi.org/10.1002/1873-3468.14940\u003c/li\u003e\n\u003cli\u003eBanker, G., \u0026amp; Goslin, K. (1998). Culturing nerve cells (2nd ed.). MIT Press.\u003c/li\u003e\n\u003cli\u003eBloem, B. R., Okun, M. S., \u0026amp; Klein, C. (2021). Parkinson\u0026rsquo;s disease. 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L-Dopa-modified microtubules lead to synapse instability in cultured neurons: Possible implications in Parkinson\u0026rsquo;s disease therapy. npj Parkinson\u0026rsquo;s Disease, 11, 298. https://doi.org/10.1038/s41531-025-01143-4\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Parkinson’s disease, L-Dopa, microtubule dynamics, tubulin tyrosination–detyrosination cycle, tubulin tyrosine ligase (TTL), vasohibin-1 (VASH1), cytoskeletal remodeling, dendritic spine stability, synaptic plasticity, dopaminergic neurotoxicity, motor fluctuations, neurodegeneration","lastPublishedDoi":"10.21203/rs.3.rs-8217923/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8217923/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eParkinson\u0026rsquo;s disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuronal loss, α-synuclein aggregation, and disrupted motor and non-motor function (Bloem et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Pharmacological replenishment of dopamine using L-3,4-dihydroxyphenylalanine (L-Dopa) remains the cornerstone of PD management; however, long-term exposure frequently induces motor fluctuations, dyskinesias, and cognitive side effects (Olanow \u0026amp; Obeso, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Recent mechanistic evidence reveals that L-Dopa itself, independent of its metabolic conversion to dopamine, can be aberrantly incorporated into neuronal microtubules, leading to structural and synaptic instability (Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). L-Dopa acts as a tyrosine analogue within the tubulin tyrosination\u0026ndash;detyrosination cycle, where tubulin tyrosine ligase (TTL) catalyzes its attachment to α-tubulin. The resulting L-Dopa\u0026ndash;modified microtubules resist enzymatic removal by the vasohibin-1/small vasohibin-binding protein (VASH1\u0026ndash;SVBP) complex, thereby accumulating in neurons and impairing cytoskeletal plasticity (Peris \u0026amp; Moutin, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zorgniotti et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Functionally, this modification disrupts dendritic spine invasion, reduces excitatory synaptic density, and perturbs intracellular transport, culminating in synaptic weakening and neurofunctional decline. These findings introduce a cytoskeletal dimension to L-Dopa neurotoxicity, linking pharmacotherapy to microtubule dysregulation and altered neuronal connectivity. Understanding the molecular interface between L-Dopa metabolism and tubulin dynamics may inform strategies to mitigate long-term treatment complications and preserve synaptic integrity in PD.\u003c/p\u003e","manuscriptTitle":"Bridging Cytoskeletal and Epitranscriptomic Mechanisms: L-DOPA–Induced Microtubule Remodeling Meets m⁶A RNA Methylation in Neural Disorders","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 16:09:45","doi":"10.21203/rs.3.rs-8217923/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":"5daa78bf-b8d7-47b2-a6fa-91e571080100","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:09:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 16:09:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8217923","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8217923","identity":"rs-8217923","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}