Sepiapterin Enhances Brain Tetrahydrobiopterin BH4-Dependent Serotonin Synthesis with Regional Specificity

Sepiapterin Enhances Brain Tetrahydrobiopterin BH4-Dependent Serotonin Synthesis with Regional Specificity | 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 Sepiapterin Enhances Brain Tetrahydrobiopterin BH4-Dependent Serotonin Synthesis with Regional Specificity Akiko Ohashi, Hiroshi Matsuoka, Shin Aizawa, Hiroyuki Hasegawa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7300477/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 Sepiapterin (SP) serves as a rate-limiting precursor of tetrahydrobiopterin (BH₄), an essential cofactor for tryptophan hydroxylase (TPH) and tyrosine hydroxylase, which are rate-limiting enzymes in the synthesis of brain monoamines. This study investigated the enhancement of brain serotonin metabolism through peripheral SP administration using comprehensive, in vitro and in vivo methodologies. In serotonin-producing RBL2H3 cells, SP exhibited a 21-fold greater cellular uptake efficiency compared to 6R-BH₄, leading to enhanced intracellular BH₄ levels and TPH activation. Kinetic analysis indicated cooperative TPH behavior (Hill coefficient = 2.1) with an apparent K M of 18.1 µM, which closely aligned with the endogenous BH₄ levels. In C57BL/6J mice, systemic SP administration demonstrated a notable biphasic dose–response pattern with a distinct threshold at approximately 20 mg/kg, delineating two mechanistically distinct regimes. Below this threshold, SP does not reach the brain; however, peripherally-generated BH₄ enters the brain parenchyma but remains predominantly extracellular, stimulating region-specific serotonin release without enhancing synthesis, manifesting as decreased 5-HIAA in brainstem serotonergic nuclei while increasing it in projection areas. Above the threshold, SP directly penetrates the blood–brain barrier and enters brain cells, elevating intracellular BH₄ levels and enhancing TPH activity, thereby producing uniform 5-HIAA increases across all brain regions. These findings establish SP as an effective strategy for enhancing brain serotonin synthesis through targeted intracellular BH₄ elevation, addressing the fundamental limitations of 6R-BH₄ supplementation that have persisted since the pioneering work of Niederwieser in the 1980s. This mechanistic breakthrough suggests a substantial therapeutic potential for various monoaminergic disorders. Cellular & Molecular Neuroscience Drug Discovery, Design, & Development Deficiency Pharmacokinetics Replenishment Sepiapterin Serotonin Tetrahydrobiopterin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Since the formulation of the serotonin hypothesis of depression in the 1960s (Coppen, 1967 ; Ross et al. , 1969), the enhancement of brain monoamine synthesis has remained a challenging therapeutic objective despite decades of extensive research. Tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH), the rate-limiting enzymes for serotonin and catecholamines, respectively, require tetrahydrobiopterin (BH 4 ), the fully reduced form of biopterin, as a cofactor (Werner et al., 2011 ). The activity of TPH adheres to Michaelis–Menten kinetics regarding BH 4 concentration; however, its availability is associated with a recycling reaction mediated by dihydropteridine reductase (DHPR), which is crucial for monoamine synthesis. BH 4 is synthesized de novo from guanosine triphosphate (GTP), with the salvage pathway providing additional protection by converting the byproduct, sepiapterin (SP), to active BH 4 (Fig. 1 ). This dual pathway system maintains intracellular BH 4 levels, which are essential for monoamine synthesis. BH 4 deficiency severely impairs monoamine synthesis, resulting in profound neurological dysfunction. The strategy of supplementing TPH and TH with BH 4 to enable these enzymes to achieve their full potential was proposed several decades ago (Niederwieser et al., 1982 ), and peripheral 6R-BH 4 administration was adopted as the standard treatment (Opladen et al., 2020 ). Nevertheless, the limited ability of 6R-BH4 to cross the blood–brain barrier (BBB) has hindered its efficacy in enhancing central serotonin synthesis (Brand et al., 1996 ). Recent studies have indicated that the administration of 6R-BH 4 stimulates the release of neurotransmitters, such as serotonin and dopamine, but with a minimal increase in transmitter synthesis (Fanet et al., 2020 ; Winn et al., 2016 ). This represents a major unmet medical need. Sepiapterin (SP) offers a potential solution as a membrane-permeable BH 4 precursor. Despite early in vitro evidence of SP-enhanced TPH activity (Hasegawa et al., 1999 ), its translation to in vivo brain applications was hindered by the prevailing view regarding rapid peripheral conversion and poor BBB penetration (Levine et al., 1987 ). Our recent research demonstrates that SP above threshold doses crosses the BBB and enters brain cells through an ENT transporter coupled with the unidirectional BH 4 salvage pathway (Ohashi et al., 2024 ) [ preprint ]. Building on this foundation, the present study investigated the downstream effects of these changes on serotonin synthesis in the brain. Based on this mechanistic insight, we systematically investigated SP-mediated enhancement of brain serotonin synthesis through: (1) in vitro validation of SP-induced BH 4 elevation and TPH activation; (2) regional analysis of 5-HT and 5-HIAA in distinct brain compartments, assuming local metabolism without inter-regional trafficking; and (3) dose–response characterization revealing biphasic effects, namely, subthreshold doses ( 20 mg/kg) produced robust increases in serotonin turnover because of SP reach. This design distinguishes the extracellular BH 4 effects from the intraneuronal SP-derived BH 4 function. Collectively, our findings establish SP as an effective strategy for enhancing central serotonin biosynthesis through targeted TPH modulation. This strategy represents a mechanistically driven intervention targeting the rate-limiting step of 5-HT synthesis. This approach addresses the urgent need for BH 4 deficiency treatment and may benefit broader neuropsychiatric conditions involving impaired monoamine synthesis. This study aimed to investigate three key hypotheses: (1) whether SP enters serotonergic neurons, (2) whether TPH activity is enhanced, and (3) whether serotonin synthesis increases above baseline. The question marks in the figure denote the key experimental targets. 2. Methods 2.1 Experimental Design Overview We used a two-phase approach: Initially, RBL2H3 cells were used to establish the mechanistic relationship between SP uptake, intracellular BH 4 elevation, and TPH activation. Subsequently, systemic SP administration in mice was used to investigate the dose-dependent effects on regional brain serotonin metabolism, focusing on TPH activity and serotonin turnover across anatomically distinct brain regions. 2.2 Materials Sepiapterin (SP) was obtained from Shiratori Pharmaceutical (Chiba, Japan). Tetrahydrobiopterin dihydrochloride (6R-BH 4 ·2HCl) was obtained from Asubio Pharma (Kobe, Japan). 7,8-Dihydrobiopterin (BH 2 ) was purchased from Schircks Laboratories (Jona, Switzerland), and N-Methylserotonin, a marker standard for high performance liquid chromatography (HPLC), and 3-hydroxybenzyl hydrazine (NSD-1015) were purchased from Nacalai Tesque (Kyoto, Japan). 2.3 Cell-Culture Experiments RBL2H3 cells (rat basophilic leukemia-derived serotonin-producing line; JCRB Cell Bank, Osaka, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37°C under 5% CO 2 . For the experiments, cells were seeded in 96-well plates (Falcon® 3072) at 10 5 cells/well and allowed to adhere overnight. To optimize TPH expression, cells were pretreated with A23187 (10 nM) for 2h, followed by 4 h incubation in "basal medium,” serum-free DMEM with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2) and 1 mM dithiothreitol (DTT) (Hasegawa et al. , 1996). BH 4 supplementation and TPH activity assessment: Reagents were refreshed by removing the previous medium, washing with Dulbecco’s phosphate buffered saline (DPBS), and replacing with a new basal medium containing the complete set of reagents. Cells were exposed to SP (0–200 µM) or 6R-BH 4 (0–1000 µM) for 30 min. To measure TPH activity, NSD-1015 (400 µM) was added for 30 min to inhibit aromatic L-amino acid decarboxylase (AADC) and enable 5-hydroxytryptophan (5HTP) accumulation. Reactions were terminated by adding an equal volume of 0.8 M perchloric acid containing 2 mM DTT, and the cells and medium were harvested. For SP/6R-BH 4 uptake measurement, basal medium without NSD-1015 was added for 30 min, followed by treatment of cells with acidic or alkaline I 2 solutions, as described in section 2.5 . 2.4 Mouse Experiments All procedures were approved by Nihon University Animal Experiment Committee (AP13M037) and complied with the ARRIVE Guidelines 2.0. Six-week-old male C57BL/6J mice (CLEA Japan, Inc., Tokyo, Japan) were housed under standard conditions (12 - h light/dark cycle, 21 – 24°C, 40%–60% humidity) with ad libitum access to the experimental diet MF (Oriental Yeast, Tokyo, Japan) and water. Experiments were conducted between 10:00 and 17:00 (Japan Standard Time; JST) after a one-week acclimation period. At the conclusion of the experiment, each mouse was sacrificed by decapitation for brain collection. Assessment of TPH activity ( Fig. 3 ): Mice (mean weight: 21.7 ± 1.2 g, n=6/group) were administered SP in saline (2 mg/mL) at a dosage of 40 mg/kg or intraperitoneal saline at t = 0, followed by NSD-1015 (5 mg/mL, 100 mg/kg) or PBS(–) administered intraperitoneally at t = 150 min. Brains were collected at t = 180 min. Dose–response study ( Fig. 4 ): Mice (mean weight: 23.9 ± 0.9 g) were administered with SP in saline (2 mg/kg) at doses of 0, 10, 20, 50 mg/kg (n=7/dose) or 125 mg/kg (n=4) intraperitoneally. Brains were harvested 6 h post-administration, when intracellular BH 4 could be selectively quantified because of the clearance of extracellular BH 4 . Brain dissection: A functional division approach was used to differentiate serotonergic cell bodies from projection territories (detailed protocol in S1. Supplementary Methods ). Briefly, the brains were dissected into three regions: Region A (brainstem with raphe nuclei), Region B (cortex/hippocampus), and Region C (subcortical structures). The coefficients of variation (CVs; mean weight ± SD mg, n = 25) were as follows: Region A, 0.062 (47.1 ± 2.9); Region B, 0.033 (242 ± 8); and Region C, 0.065 (90.3 ± 5.9). This approach enables a quantitative assessment of the regional serotonin synthesis capacity. Tissue processing: Frozen tissues were homogenized using a mechanical homogenizer (T 25-S1, IKA Labortechnik, Breisgau, Germany) on ice in 0.1 M HCl containing 0.5 µM N-methylserotonin (internal standard) and aliquoted for biochemical analyses. 2.5 Biochemical Determinations Biopterin measurements: The Fukushima–Nixon differential oxidation method was used for biopterin quantification (Fukushima et al. , 1980). This technique provides stable and reproducible results for large sample sizes. In this method, acidic I 2 oxidation yields total reduced biopterins, BH 4 , biopterin-4a-carbinilamine, quinonoid dihydrobiopterin (qBH 2 ), and BH 2 , whereas alkaline I 2 oxidation yields only BH 2 . In this manuscript, "BH 4 " represents physiologically relevant cofactor levels, although measurements represent total reduced biopterins (tBH 4 ) from acid oxidation alone, thereby avoiding calculation errors from subtraction. Sample preparation: For cultured cells ( Fig . 2 ), the cells in the wells were washed with DPBS and directly treated with 50 µL iodine solution. For brain tissue ( Fig . 4 ), frozen homogenates (50 µL aliquots in 0.1 M HCl) were thawed on ice and mixed with an equal volume of iodine solution. All procedures were performed under dim light to avoid photodegradation. Oxidation procedure: Samples were treated with acidic I₂ (2% I₂, 3% KI in 0.1 M HCl) or alkaline I₂ (2% I₂, 3% KI in 0.2 M NaOH), incubated at 30°C for 60 min, and then quenched with an equal volume of 0.2 M ascorbic acid in 1 M perchloric acid. After centrifugation (9,100 × g, 10 min, 4°C), the supernatants were analyzed using HPLC (octadecylsilica (ODS) column: JASCO Finepak SIL C18T-5, 40˚C, 7% methanol, fluorescence detection: E x = 350, E m = 450 nm). A mixture of authentic biopterin and 2-amino-4-hydroxypteridine (pterin) was run as an external standard every 10 analyses. Rationale for tBH 4 reporting: In the TPH-DHPR coupled system, BH 4 and qBH 2 were dynamically partitioned between enzymes. DHFR in the BH 4 salvage pathway rescues most of the BH 2 . Reporting acid oxidation values (tBH 4 ) reflects the physiologically relevant cofactor pool without introducing any subtraction errors. 5HTP, 5-HT, and 5-HIAA: The indole compounds were quantified using HPLC with native fluorescence detection as previously described (Hasegawa et al. , 1987), with minor modifications specific to 5HTP measurement in brain tissue. Culture cells or tissue homogenates were mixed with perchloric acid/DTT solution (final concentrations: 0.4 M and 1 mM). After centrifugation (9,100 × g , 10 min, 4 °C), the supernatant was analyzed using an ODS column (JASCO Finepak SIL C18T-5, 40 °C) with fluorescence detection ( E x: 302 nm, E m: 350 nm). The mobile phase consisted of 10 mM sodium acetate (pH 3.5, adjusted with formic acid), acetonitrile, and methanol (100:5:7, v/v/v). External standards containing tryptophan, 5-HTP, 5-HT, 5-HIAA, and N-methylserotonin were run after every 10 samples. Specifically, for the in situ TPH activity assay ( Fig. 3c ), the mobile phase was modified to a 100:9:1.5 mixture to separate 5HTP from brain-specific contaminants. 2.6 Statistical Analysis Data are presented as mean ± SEM with individual data points. Statistical analyses were performed using Excel with the Bell Curve add-in (Social Research Information; Tokyo, Japan). Two-way analysis of variance (ANOVA) with Dunnett's test was used for the in vitro dose–response analysis ( Fig. 2d ). Multi-way ANOVA with Tukey's post-hoc comparisons was applied to the in vivo experiments ( Figs. 3–4 ). For the kinetic analysis in Fig. 2 , TPH activity data were fitted to both simple ( n = 1) and extended ( n > 1) Michaelis–Menten equations using non-linear regression. The extended equation is v = V max ·[ S ] ⁿ /((app K M ) ⁿ + [ S ] ⁿ ) where v is the reaction velocity, [ S ] is the substrate concentration (tBH 4 ), n is the Hill coefficient, and app K M is the apparent Michaelis constant. For Fig. 4 , raw data analyses ( upper panels) determined statistical significance, with effect sizes calculated using Hedges' g . Normalized data ( lower panels) show the same datasets as fold-changes, preserving statistical significance while facilitating the interpretation of relative changes across regions. Linear regression was used to determine the threshold doses from the x-intercepts. Effect size interpretations followed Cohen's conventions: η 2 (0.01 = small, 0.06 = medium, 0.14 = large) and Hedges' g (0.2 = small, 0.5 = medium, 0.8 = large). Post-hoc power analysis was conducted for the dose–response study. For the 125 mg/kg group (n=4), with the observed effect size (Cohen's d = 1.33), the achieved power was 0.47. Sample size calculations indicated that n=9 per group would achieve 80% power for detecting similar effect sizes. 3. Results 3.1 Cellular Uptake of SP/6R-BH 4 and Their Effects on TPH Activity The mechanistic relationship between extracellular SP/6R-BH 4 exposure and intracellular TPH activation was examined using serotonin-producing RBL2H3 cells. Cells were exposed to varying concentrations of SP (0–200 µM) or 6R-BH 4 (0–1000 µM) for 30 min, followed by the measurement of intracellular BH 4 levels (tBH 4 ) and TPH activity (Fig. 2 ). SP demonstrated markedly superior cellular uptake compared to that of 6R-BH 4 . Linear regression analysis revealed uptake rates of 0.66 ± 0.01 pmol/well per µM for SP ( R ² = 0.993, n = 32) vs. 0.031 ± 0.001 pmol/well per µM for 6R-BH 4 ( R ² = 0.937, n = 48), representing a 21-fold difference. This differential uptake efficiency is attributed to the substrate specificity of the ENT2 transporter and the subsequent conversion of SP to BH 4 via the salvage pathway (Ohashi et al., 2011 ; Sawabe et al., 2008 ). Enhancement of TPH activity exhibited distinct concentration-response patterns: SP achieved maximum activation at 30–50 µM extracellular concentration, whereas 6R-BH 4 required > 500 µM for comparable effects. To achieve a 25% increase in TPH activity over the baseline, approximately 0.1 µM SP or 3 µM 6R-BH 4 was required. Notably, when TPH activity was plotted against the achieved intracellular BH 4 levels rather than extracellular exposure concentrations, the data from both treatments converged onto a single curve (Fig. 2 e). This convergence indicates that TPH activation is solely dependent on intracellular BH 4 levels, with enhanced cellular uptake being entirely responsible for the superior efficacy of SP. The cellular TPH system operates as a coupled enzyme complex, wherein TPH activity is intricately associated with BH 4 recycling via DHPR. An analysis of the TPH reaction velocity in relation to total intracellular total BH 4 (tBH 4 = BH 4 + qBH 2 + biopterin-4a-carbinolamine) revealed cooperative kinetics fitting the extended Michaelis–Menten equation: \(\:v=\frac{{V}_{\text{m}\text{a}\text{x}}\cdot\:{\left[S\right]}^{n}}{{{\text{a}\text{p}\text{p}K}_{\text{M}}}^{n}+{\left[S\right]}^{n}}\) …… (1). Non-linear regression yielded n = 2.1, V max = 40.0 pmol/30-min/well, and app K M = 18.1 µM ( R ² = 0.906, n = 81). The extended model provided a significantly better fit compared to simple Michaelis–Menten kinetics ( n = 1; R ² = 0.848), confirming cooperative substrate binding. The Hill coefficient of 2.1 suggests positive cooperativity within the TPH-DHPR complex. Notably, app K M approximated the endogenous tBH 4 concentration (17.9 ± 0.5 µM, estimated using standard assumptions of 1.80 pL spherical cell volume and 1×10⁵ cells/well, Fig. 2 c), suggesting that cells maintain BH 4 levels near the K M for optimal responsiveness to cofactor fluctuations. This coupling creates a fundamental constraint: since v TPH = v DHPR in the recycling system, and [BH 4 ] + [qBH 2 ] = [tBH 4 ]. Hence, the available BH 4 for TPH is always less than the total tBH 4 . As such, DHPR both sustains and limits TPH activity, explaining why, even in the presence of high BH 4 concentrations, cellular systems cannot reach the theoretical V max of either enzyme. 3.2 TPH activation in vivo is accompanied by increased 5-HIAA across brain regions To establish a region-specific analysis of SP effects on serotonin metabolism, TPH activity was assessed 2.5 h following intraperitoneal SP administration (40 mg/kg) using metabolic arrest with NSD-1015 (Fig. 3 ). The brains were divided into three regions based on serotonergic anatomy: brainstem containing cell bodies (Region A, 47.1 ± 2.9 mg), cortex/hippocampus with projections (Region B, 242 ± 8 mg), and the remaining forebrain (Region C, 90.3 ± 5.9 mg). Baseline TPH activity exhibited pronounced regional heterogeneity (ANOVA: F (2,22) = 285, p < 0.001), with Regions B and C exhibiting approximately twice the activity of Region A (ratio 1:1.81:1.94), reflecting axonal TPH enzyme transport from the brainstem cell bodies. SP administration enhanced TPH activity differentially across regions ( F (1,11) = 6.23, p = 0.030), with significant increases in Regions A (+ 12%) and C (+ 16%, both p < 0.001). SP-induced distinct patterns of 5-HIAA changes: Region B exhibited the most pronounced increase (+ 67%, p < 0.001), while 5-HIAA/5-HT ratios increased significantly across all regions (A: +23%, B: +67%, C: +31%; all p < 0.001). These differential responses demonstrate that (1) brain regions must be analyzed separately because of distinct TPH distribution patterns and (2) concurrent 5-HIAA measurement is essential for evaluating serotonin turnover. This single-dose study established a methodological framework for a comprehensive dose–response analysis required to elucidate the region-specific effects of SP on serotonin metabolism. Subsequently, we conducted comprehensive dose–response studies across multiple brain regions. 3.3 Dose-Dependent Effects of SP on Brain BH 4 and Serotonin Metabolism Systemic SP administration (0–125 mg/kg) intraperitoneally induced dose-dependent changes in brain BH 4 and monoamine metabolism at 6 h post-injection (Fig. 4 , organized in matrix format for systematic regional comparisons), when intracellular BH 4 can be selectively quantified owing to the clearance of extracellular BH 4 (Ohashi et al., 2024 ) [ preprint ]. To evaluate these effects across brain regions with different baseline concentrations, we analyzed both the absolute amounts ( upper panels) and fold-changes from the baseline ( lower panels). Absolute measurements revealed dose-dependent increases in total brain BH 4 ( Fig. 4 b 1 ), reaching a 3.4-fold elevation at 125 mg/kg (ANOVA: F (4,26) = 15.3, p < 0.001). 5-HT levels showed modest variations ( Fig. 4 c 1 ; F (4,26) = 1.69, p = 0.184), whereas 5-HIAA exhibited significant changes ( Fig. 4 d 1 ; F (4,26) = 6.97, p < 0.001, η ² = 0.517, very large effect). Given the substantial differences in baseline concentrations across regions (Fig. 4 , leftmost columns), we normalized the data to the baseline values. This normalization revealed distinct responses, suggesting that the threshold effect of the SP dose was masked by baseline differences. This convergent threshold suggests a systemic barrier to BH 4 elevation at lower doses. The dose-dependent changes in effect sizes, from negative values at 10–20 mg/kg (Hedges' g = − 0.58 to − 0.28) to moderate positive effects at 50 mg/kg ( g = 0.442) and large effects at 125 mg/kg ( g = 0.758), demonstrate a clear threshold phenomenon. Despite moderate statistical power at 125 mg/kg (1 − β = 0.47, n = 4), the large effect size confirms robust biological activity above the threshold dose, consistent with the ~ 20 mg/kg threshold determined by linear regression and BH₄-mediated TPH activation. BH 4 responses: Brain BH 4 exhibited a biphasic dose-dependency (Fig. 4 b). No significant increases were observed below 20 mg/kg in any of the regions. Normalized BH 4 responses displayed remarkable linearity above the threshold SP dose, which was consistent across regions A, B, and C, with regression lines intercepting the x-axis at 17.8 ( R 2 = 0.69), 11.7 ( R 2 = 0.77), and 11.45 ( R 2 = 0.81) mg/kg, respectively (Fig. 4 b 2 –b 5 , dashed lines). The baseline BH 4 abundance in Region B was 3-fold higher than that in Region A (Fig. 4 b 2 ), consistent with the 1.81-fold higher TPH activity (Fig. 3 c). 5-HT levels : Regional analysis revealed heterogeneous responses (multi-way ANOVA: F (2,52) = 5.49, p = 0.007). Region A exhibited biphasic changes, with significant decreases at 10–50 mg/kg, followed by an elevation at 125 mg/kg ( p < 0.01). Region B maintained stable 5-HT levels throughout, whereas Region C displayed modest elevation trends at high doses. 5-HIAA patterns : Two distinct dose-dependent patterns emerged, demarcated by the BH 4 threshold (Fig. 4 d). Below the threshold, contrasting regional responses occurred: Region A showed decreased 5-HIAA ( p = 0.049), while Region B showed an increase ( p = 0.025), with no net whole-brain change because of Region B's larger tissue mass. Above the threshold, all regions exhibited dose-dependent 5-HIAA increases (Region A: p < 0.01, Region B: p < 0.01, Region C: p < 0.05), accompanied by maintained or increased 5-HT levels, thereby demonstrating a successful enhancement of 5-HT synthesis. These biphasic 5-HIAA responses indicate two distinct mechanisms: below the threshold, peripherally-generated BH 4 induces region-specific monoamine release without enhancing its synthesis. Above this threshold, SP-derived intracellular BH 4 enhances TPH activity, increasing both synthesis and turnover. This dual mechanism explains the complex dose–response relationships and establishes the optimal dosing requirements for therapeutic serotonin enhancement. 4. Discussion This study revealed that peripheral sepiapterin (SP) enhances brain serotonin synthesis through a defined route: crossing the BBB, entering neurons via transporters, increasing BH 4 levels, and activating TPH. Rather than data-driven screening, this is a hypothesis-driven intervention targeting a known biosynthetic bottleneck. Our results revealed clear region- and dose-dependent associations between SP and BH 4 elevation and the serotonin turnover. A summary of the model is presented in Fig. 5 . 4.1 Mechanistic Interpretation at the Cellular Level Our cellular studies revealed that SP represents a superior strategy for enhancing brain serotonin synthesis compared to direct BH 4 supplementation. Using intact RBL2H3 cells, which share key functional properties with serotonergic neurons that project throughout the brain, including serotonin production, storage, and release, we preserved cellular integrity to clarify the critical role of precursor transport in TPH enhancement. The observed superior uptake efficiency of SP compared to that of 6R-BH 4 reflects the substrate specificity of ENT2 (6R-BH 4 :BH 2 :SP ratio = 1:1.92:15.6) and the synergistic effect of the push–pull drive of SP through the salvage pathway (Sawabe et al., 2008 ). The key transporter, ENT2, is abundant throughout the brain parenchyma (Lu et al., 2004 ). This differential transport efficiency translates directly to enhanced TPH activation (Fig. 2 ): achieving equivalent intracellular BH 4 levels requires impractically higher 6R-BH 4 exposure. The convergence of SP and 6R-BH 4 data when plotted against intracellular BH 4 concentrations confirms that precursors function equivalently as coenzymes once internalized, with efficacy differences explained solely by uptake efficiency, which in turn confirms that extracellular BH 4 remains extracellular and is not efficiently taken up into the cells. The obligate coupling between TPH and DHPR activities creates unique kinetic constraints within the cellular system. Our analysis revealed cooperative kinetics (Hill coefficient = 2.1) with an app K M of 18.1 µM, which was remarkably close to the endogenous tBH 4 levels (17.9 µM). This alignment suggests that cells maintain BH 4 near the " K M level" for optimal responsiveness, consistent with previous findings that intracerebroventricular 6R-BH 4 can double brain TPH activity, indicating subsaturating baseline BH 4 levels (Miwa et al., 1985 ). BH 2 , which is derailed from the BH 4 redox cycle by spontaneous tautomerization (qBH 2 →BH 2 , T 1/2 = 1.6 min (Watabe, 1978 )), is not recycled by DHPR. Instead, it is primarily recovered via the salvage pathway, and the remaining portion leaks out of the cell (permeability BH 2 > BH 4 ), resulting in substantial BH 4 loss. This loss of cellular BH 4 is compensated by de novo synthesis. Critically, the recycling of BH 4 between TPH and DHPR ensures a stable supply of BH 4 , but limits the available BH 4 to less than the total tBH 4 . A weak DHPR increases qBH2 in the equation [BH 4 ] = [tBH 4 ] - [qBH 2 ]. Regardless of the concentration of tBH 4 , the resulting TPH activity will be lower than its potential activity, that is, app V max < V max,TPH . This explains why DHPR deficiency impairs monoamine production despite significant BH 4 levels being maintained (Xu et al., 2014 ), which is a key insight for understanding inherited metabolic disorders. Although specific regulatory details may differ between RBL2H3 cells and authentic serotonergic neurons, these fundamental characteristics of BH 4 -dependent hydroxylase systems are likely to be broadly applicable. 4.2 Regional Characteristics of the Serotonergic System at Baseline Regional analysis has provided fundamental insights into the anatomical and functional organization of serotonin production in the brain. Previous studies have shown that GTPCH1 is primarily expressed in serotonergic neurons, with a lesser but significant contribution from catecholaminergic neurons (Hirayama et al. , 1998; Lentz et al. , 1996). The baseline tBH 4 distribution shown in Fig. 4 b 2 aligned with the GTPCH1 expression pattern. It is highest in Region A containing serotonergic cell bodies, intermediate in Region C with mixed serotonergic projections and dopaminergic neurons, and lowest concentration in Region B. Although the tBH 4 concentration in Region B is approximately half that in Region A, its tBH 4 content (nmol/g × g) is three times greater because of the larger tissue volume. Based on the empirical consistency of intracellular BH 4 being close to app K M (approximately 18 µM ≈ 1.8 nmol/g), the tBH 4 concentration in Region A (0.82 nmol/g) corresponds to 2.1 mg of cytoplasm containing 1.8 nmol/g tBH 4 , which represents 4.4% of the tissue mass (47.1 mg). Presumably, this represents the cytoplasmic volume of serotonergic neurons in the brainstem. Similarly, 6.52 mg of cytoplasm maintained TPH activity in Region B, corresponding to 2.7% of the tissue mass (242 mg). The baseline activity of TPH is maintained by the supply of consumed BH 4 via DHPR recycling. Specifically, the TPH activity per tBH 4 concentration, that is, the cofactor utilization rate, was lower in region B than in Region A (compare Fig. 3 c with Fig. 4 b 2 ). As efficient cofactor utilization is determined by the DHPR/TPH ratio via the partitioning of tBH 4 to BH 4 and qBH 2 , it is unclear whether this regional difference is because of TPH or DHPR insufficiency. This quantitative demonstration that axonal varicosities and synaptic terminals in projection regions (Region B) actively produce serotonin challenges the traditional view of limiting synthesis to cell body regions. These baseline characteristics establish the foundation for understanding the differential regional effects of SP and highlight the importance of analyzing both synthesis capacity and metabolic turnover across anatomically distinct serotonergic compartments. 4.3 Biphasic Response Mechanisms The threshold phenomenon at ~ 20 mg/kg SP represents a pivotal discovery that explains the previously inconsistent findings regarding SP efficacy. This threshold reflects the competition between peripheral and central SP uptake, with peripheral organs preferentially sequestering SP at low doses. Low-dose mechanism (< 20 mg/kg) : Below this threshold, SP is rapidly converted to BH 4 in peripheral organs, with subsequent systemic BH 4 delivery to the brain, but is largely cleared after 6 h (Ohashi et al., 2024 ) [ preprint ]. This peripherally-derived BH 4 , while entering the brain parenchyma, remains predominantly extracellular and triggers monoamine release without enhancing monoamine synthesis—consistent with the findings of previous 6R-BH 4 studies showing release stimulation independent of synthesis promotion (Fanet et al., 2020 ; Koshimura et al., 1994 ; Winn et al., 2016 ). The contrasting regional 5-HIAA responses (decreased in Region A increased in Region B) likely reflect the differential expression of serotonin autoreceptors: somatodendritic 5-HT1A receptors in Region A providing inhibitory feedback versus terminal 5-HT1B receptors in Region B modulating release dynamics. This explains why cellular studies show linear SP responses (Fig. 2 ), whereas in vivo responses exhibit threshold behavior. High-dose mechanism (> 20 mg/kg) Above this threshold, peripheral SP uptake saturates, allowing systemic SP circulation and brain delivery. SP crosses the BBB, enters neurons via ENT2 transporters, and is converted to intracellular BH 4 via the salvage pathway. This intracellular BH 4 enhancement follows kinetics similar to those of cultured cells (Fig. 2 e), with TPH activity increasing according to cellular BH 4 levels. Notably, the regional heterogeneity observed at low doses was reversed at high doses, producing uniform serotonin parameter increases across all regions. This suggests that enhanced intracellular BH 4 -driven synthesis overwhelms the region-specific release modulation observed at low doses, providing a unified mechanism for therapeutic serotonin enhancement in diverse brain regions. 4.4 Comparison with Existing Research and Mechanistic Implications Our discovery of the biphasic dose–response pattern of SP provides a mechanistic framework for addressing four decades of challenges in BH 4 -based therapeutics. Since Niederwieser et al.’s seminal work in the 1980s (Curtius et al., 1983 ; Niederwieser et al., 1982 ), 6R-BH 4 has been the standard for BH 4 replacement, despite demonstrating inferior cellular uptake compared to SP (Hasegawa et al., 2005 ; Sawabe et al., 2008 ). The persistent use of 6R-BH 4 in the field has overlooked a critical distinction: while 6R-BH 4 increases brain BH 4 levels, this BH 4 remains predominantly extracellular, stimulating monoamine release without enhancing its synthesis (Koshimura et al., 1994 ). This explains why congenital BH 4 deficiency syndromes require combination therapy with L-DOPA and 5HTP, rather than 6R-BH 4 monotherapy (Opladen et al., 2020 ). However, to avoid adverse effects, including diarrhea, tremors, and hypothermia, 5HTP must be administered slowly and continuously (Jacobsen et al., 2016 ). These adverse effects likely result from bypassing the intrinsic regulatory function of TPH, which provides a finely tuned 5HTP supply according to demand. Our threshold phenomenon (~ 20 mg/kg in mice) represents an inflection point whereby SP dosage transitions from peripheral BH 4 production (release effect only) to direct SP delivery to brain cells (enabling synthesis enhancement). Understanding this mechanism rationalizes previous BH 4 -related findings and represents a paradigm shift from empirical trial-and-error to rational, mechanism-based BH 4 replacement. Implications for specific BH 4 deficiencies: SP administration requires intact salvage pathway enzyme activity. In SPR deficiency, SP cannot be converted to BH 4 , rendering it ineffective. Although alternative pathways (carbonyl reductase and DHFR) provide limited BH 4 in the liver, their brain activity remains unclear (Bonafe et al., 2001 ). DHPR deficiency presents a different challenge, as it impairs BH 4 utilization rather than production. BH 4 utilization requires functional DHPR. Paradoxically, DHPR-knockout mice exhibit significant levels of brain BH 4 (Xu et al., 2014 ), confirming that BH 4 availability alone is insufficient without functional recycling. These mechanistic constraints define patient populations that would not benefit from SP therapy, underscoring the importance of enzymatic profiling before initiating treatment. 4.5 Clinical Significance and Therapeutic Potential The enhancement of brain BH 4 mediated by SP presents unique advantages for treating monoaminergic disorders. By elevating intraneuronal BH 4 levels above threshold levels, SP simultaneously enhances the synthesis of all monoamines—serotonin, dopamine, and norepinephrine—because of their common dependence on BH 4 -dependent hydroxylases. This multi-system approach contrasts with current single-target strategies, such as selective serotonin reuptake inhibitors (SSRIs), and may more adequately address complex disorders, such as major depression, which involves multiple neurotransmitter dysregulations. Our approach addresses a fundamental limitation of current therapies: while SSRIs increase synaptic serotonin (5-HT ext ), they cannot overcome the reduced synthesis capacity in individuals with diminished TPH activity. Although 5-HT ext is only approximately 1/10,000th of the 5-HT in tissues, its turnover is extremely rapid, making its total traffic comparable to that of released vesicles. Its sustained release is limited by the size of the vesicular pool and, therefore, the capacity to produce 5-HT (Calcagno et al., 2007 ). Furthermore, this pool is maintained by rapid turnover throughout the brain as a single compartment with T 1/2 ≈ 1 h (Carlsson et al., 1972 ). By enhancing TPH activity while preserving enzymatic regulatory mechanisms, SP increases serotonin production capacity, providing resilience against depletion during high-demand states (e.g., stress). For other conditions, the biphasic response pattern facilitates personalized medicine approaches: low-dose (monoamine release) versus high-dose (enhanced synthesis) can be tailored to specific pathophysiological conditions. Regional heterogeneity at low doses versus uniform enhancement at high doses suggests the potential for either targeted or global therapeutic strategies, depending on clinical needs. 4.6 Limitations and Future Directions However, some limitations must be considered before our findings can be clinically applied. Pharmacokinetic optimization : Our single-dose studies revealed rapid SP clearance (urinary excretion within 1 h; (Ohashi et al., 2024 ) [ preprint ]). Future studies should explore repeated dosing strategies to maintain therapeutic brain BH 4 levels. Route-dependent thresholds : The ~ 20 mg/kg threshold (mouse, intraperitoneal) may differ with alternative administration routes. Human translation requires comprehensive pharmacokinetic/pharmacodynamic studies that involve multiple delivery methods. Catecholaminergic effects : Although we focused on serotonergic parameters, the effects of SP on dopamine and norepinephrine synthesis via TH enhancement remain uncharacterized. Functional outcomes : Behavioral and cognitive consequences of SP-induced neurochemical changes require systematic evaluation using validated paradigms relevant to monoaminergic function. Drug interactions : Potential synergistic or antagonistic effects with existing monoaminergic therapies (SSRIs, MAO inhibitors) require investigation before clinical application. Addressing these limitations will refine dosing strategies, identify optimal patient populations, and establish the position of SP within existing therapeutic frameworks for monoaminergic disorders. Abbreviations 5-HT, serotonin; 5-HIAA, 5-hydroxy-indoleacetic acid; 5HTP, 5-hydroxy-L-tryptophan; 6R-BH 4 , 6R-tetrahydrobiopterin; AADC, aromatic-L-amino acid decarboxylase; BBB, blood–brain barrier; BH 2 , 7,8-dihydrobiopterin; BH 4 , tetrahydrobiopterin; DHFR, dihydrofolate reductase; DHPR, dihydropteridine reductase; MAO, monoamine oxidase; qBH 2 , quinonoid dihydrobiopterin; SP, sepiapterin; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase Declarations Conflict of Interest Statement H.H. declares no current conflicts of interest. In the past, H.H. served as an advisor to Shiratori Pharmaceutical Co., Ltd (2013), was a member of advisory board for Censa Pharmaceuticals Inc. (2016–2021), and holds stock options in Censa Pharmaceuticals Inc. (now acquired by PTC Therapeutics). Intellectual property rights in PCT application WO2011/132435, invented by H.H. and S.A. were assigned to Censa Pharmaceuticals Inc. (acquired by PTC Therapeutics). A.O., H.M. and S.A. declare no conflict of interest. Author Contributions A.O. and H.H.: Conceptualization, Methodology, Investigation, Formal analysis, Writing - Original Draft, Visualization; H.M.: Formal analysis, Validation; S.A.: Resources, Project administration, Funding acquisition. All authors reviewed and approved the final manuscript. Acknowledgements This research was supported by Shiratori Pharmaceutical Co., Ltd. (2013), and the Dental Research Center, Nihon University School of Dentistry (DRC(B)-2023-1, DRC(B)-2024-1). We are grateful to Prof. Kazuhiro Nakamura of the Applied Health Sciences Unit, Department of Medical Laboratory Science, Gunma University, for his valuable advice regarding the brain dissection protocol. We also thank Prof. Tomihisa Takahashi of the Department of Anatomy, Nihon University School of Dentistry, and Dr. Tomonori Harada of the Department of Anatomy and Functional Morphology, Nihon University School of Medicine, for their kind support of this research. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Bonafe, L., Thony, B., Penzien, J. M., Czarnecki, B., & Blau, N. (2001). "Mutations in the sepiapterin reductase gene cause a novel tetrahydrobiopterin-dependent monoamine-neurotransmitter deficiency without hyperphenylalaninemia." Am. J. Hum. Genet., 69(2): 269-277. Brand, M. P., Hyland, K., Engle, T., Smith, I., & Heales, S. J. (1996). "Neurochemical effects following peripheral administration of tetrahydropterin derivatives to the hph-1 mouse." J. 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Dispos., 32(12): 1455-1461. doi:10.1124/dmd.104.001123 Miwa, S., Watanabe, Y., & Hayaishi, O. (1985). "6R-L-erythro-5,6,7,8-tetrahydrobiopterin as a regulator of dopamine and serotonin biosynthesis in the rat brain." Arch. Biochem. Biophys., 239(1): 234-241. doi:0003-9861(85)90831-8 [pii] Niederwieser, A., Curtius, H. C., Wang, M., & Leupold, D. (1982). "Atypical phenylketonuria with defective biopterin metabolism. Monotherapy with tetrahydrobiopterin or sepiapterin, screening and study of biosynthesis in man." Eur. J. Pediatr., 138(2): 110-112. Ohashi, A., Matsuoka, H., Nakamaru-Ogiso, E., Aizawa, S., & Hasegawa, H. (2024). "Peripheral Administration of Sepiapterin Replenishes Brain Tetrahydrobiopterin." Research Square [Preprint]. doi:10.21203/rs.3.rs-4111864/v2 Ohashi, A., Sugawara, Y., Mamada, K., Harada, Y., Sumi, T., Anzai, N., Aizawa, S., & Hasegawa, H. (2011). "Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters: A plausible gateway for the salvage pathway of tetrahydrobiopterin biosynthesis." Mol. Genet. Metab., 102(1): 18-28. doi:10.1016/j.ymgme.2010.09.005 Opladen, T., Lopez-Laso, E., Cortes-Saladelafont, E., Pearson, T. S., Sivri, H. S., Yildiz, Y., Assmann, B., Kurian, M. A., Leuzzi, V., Heales, S., Pope, S., Porta, F., Garcia-Cazorla, A., Honzik, T., Pons, R., Regal, L., Goez, H., Artuch, R., Hoffmann, G. F., Horvath, G., Thony, B., Scholl-Burgi, S., Burlina, A., Verbeek, M. M., Mastrangelo, M., Friedman, J., Wassenberg, T., Jeltsch, K., Kulhanek, J., Kuseyri Hubschmann, O., & International Working Group on Neurotransmitter related, D. (2020). "Consensus guideline for the diagnosis and treatment of tetrahydrobiopterin (BH4) deficiencies." Orphanet J. Rare Dis., 15(1): 126. doi:10.1186/s13023-020-01379-8 Ross, S. B., & Renyi, A. L. (1969). "Inhibition of the uptake of tritiated 5-hydroxytryptamine in brain tissue." Eur. J. Pharmacol., 7(3): 270-277. doi:10.1016/0014-2999(69)90091-0 Sawabe, K., Yamamoto, K., Harada, Y., Ohashi, A., Sugawara, Y., Matsuoka, H., & Hasegawa, H. (2008). "Cellular uptake of sepiapterin and push-pull accumulation of tetrahydrobiopterin." Mol. Genet. Metab., 94(4): 410-416. doi:10.1016/j.ymgme.2008.04.007 Watabe, S. (1978). "Purification and characterization of tetrahydrofolate•protein complex in bovine liver." J. Biol. Chem., 253(19): 6673-6679. Werner, E. R., Blau, N., & Thöny, B. (2011). "Tetrahydrobiopterin: Biochemistry and pathophysiology." Biochem. J., 438(3): 397-414. doi:doi: 10.1042/BJ20110293 Winn, S. R., Scherer, T., Thöny, B., & Harding, C. O. (2016). "High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU)." Mol. Genet. Metab., 117(1): 5-11. doi:10.1016/j.ymgme.2015.11.012 Xu, F., Sudo, Y., Sanechika, S., Yamashita, J., Shimaguchi, S., Honda, S., Sumi-Ichinose, C., Mori-Kojima, M., Nakata, R., Furuta, T., Sakurai, M., Sugimoto, M., Soga, T., Kondo, K., & Ichinose, H. (2014). "Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse." FEBS Lett., 588(21): 3924-3931. doi:10.1016/j.febslet.2014.09.004 Additional Declarations The authors declare no competing interests. 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. 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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-7300477","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":496030360,"identity":"61fd4486-ab9c-4187-9702-d5f520800511","order_by":0,"name":"Akiko Ohashi","email":"","orcid":"","institution":"Nihon University School of Dentistry, Tokyo, Japan; Nihon University School of Medicine, Tokyo, Japan","correspondingAuthor":false,"prefix":"","firstName":"Akiko","middleName":"","lastName":"Ohashi","suffix":""},{"id":496038509,"identity":"98c149ed-1099-4e7d-8b73-976664e663ba","order_by":1,"name":"Hiroshi Matsuoka","email":"","orcid":"","institution":"Teikyo University of Science, Tokyo, Japan","correspondingAuthor":false,"prefix":"","firstName":"Hiroshi","middleName":"","lastName":"Matsuoka","suffix":""},{"id":496038510,"identity":"c4ecab93-aca9-4044-b2e1-bd0e465b54fc","order_by":2,"name":"Shin Aizawa","email":"","orcid":"","institution":"Nihon University School of Medine, Tokyo, Japan","correspondingAuthor":false,"prefix":"","firstName":"Shin","middleName":"","lastName":"Aizawa","suffix":""},{"id":496038511,"identity":"22c60326-2a89-4915-8a50-c782613486fe","order_by":3,"name":"Hiroyuki Hasegawa","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-0942-6481","institution":"Nihon University School of Medicine, Tokyo, Japan","correspondingAuthor":true,"prefix":"","firstName":"Hiroyuki","middleName":"","lastName":"Hasegawa","suffix":""}],"badges":[],"createdAt":"2025-08-05 12:13:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7300477/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7300477/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88751133,"identity":"9d123826-ba81-49bf-89af-7bc01d0f41d6","added_by":"auto","created_at":"2025-08-11 06:14:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":327649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTetrahydrobiopterin and its role in serotonin biosynthesis.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Sepiapterin (SP) is a reduced pterin with a three-carbon side chain. (\u003cstrong\u003eb\u003c/strong\u003e) SP serves as a membrane-permeable precursor of tetrahydrobiopterin (BH\u003csub\u003e4\u003c/sub\u003e), which is converted via the BH\u003csub\u003e4\u003c/sub\u003e salvage pathway. BH\u003csub\u003e4\u003c/sub\u003e is an essential cofactor for tryptophan hydroxylase, which is the rate-limiting enzyme in serotonin (5-HT) synthesis. Our previous research demonstrated that high-dose SP (\u0026gt;20 mg/kg) traverses the BBB and elevates BH\u003csub\u003e4\u003c/sub\u003e levels in brain cells (Ohashi\u003cem\u003e et al.\u003c/em\u003e, 2024))\u003cstrong\u003e \u003c/strong\u003e[\u003cstrong\u003epreprint\u003c/strong\u003e], although it was not determined whether serotonergic neurons were among these cells.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7300477/v1/72c2dfe7350d3f7d6adfaacd.png"},{"id":88751135,"identity":"e009a96d-f954-487d-a3eb-6f6b07084399","added_by":"auto","created_at":"2025-08-11 06:14:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":306183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential uptake of sepiapterin versus 6R-BH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e and resultant enhancement of TPH activity––Concentration-dependent effects and mechanistic analysis in RBL2H3 cells.\u003c/strong\u003e\u003cem\u003e \u003c/em\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Experimental timeline: RBL2H3 cells were exposed to A23187 (10 nM, 2 h), followed by a 4-h incubation period. Cells were subsequently treated with SP or 6R-BH\u003csub\u003e4\u003c/sub\u003e for 30 min, and TPH activity was assessed using NSD-1015 (400 µM for 30 min). (\u003cstrong\u003eb\u003c/strong\u003e) Schematic representation of the TPH assay by measuring 5HTP accumulation with NSD-1015. (\u003cstrong\u003ec\u003c/strong\u003e) Intracellular BH\u003csub\u003e4\u003c/sub\u003e levels after exposure to SP (0–200 µM, \u003cem\u003ered\u003c/em\u003e) for 30 min or 6R-BH\u003csub\u003e4\u003c/sub\u003e (0–1000 µM, \u003cem\u003eblue\u003c/em\u003e). Baseline BH\u003csub\u003e4\u003c/sub\u003e: 3.23 ± 0.10 pmol/well (n=8). (\u003cstrong\u003ed\u003c/strong\u003e) TPH activity measured as 5HTP formation after exposure to SP (0–200 µM) and 6R-BH\u003csub\u003e4\u003c/sub\u003e (0–500 µM). Baseline TPH activity: 19.4 ± 0.6 pmol/30 min/well (n=12). (\u003cstrong\u003ee\u003c/strong\u003e) Correlation between intracellular BH\u003csub\u003e4\u003c/sub\u003e concentration and TPH activity. Data from both SP and 6R-BH\u003csub\u003e4\u003c/sub\u003e treatments converged when plotted against the achieved intracellular BH\u003csub\u003e4\u003c/sub\u003e levels. \u003cem\u003eSolid\u003c/em\u003e line: cooperative kinetic model (\u003cem\u003en\u003c/em\u003e = 2.1); dotted line: simple Michaelis–Menten model (\u003cem\u003en\u003c/em\u003e = 1). Refer to the Results section “Apparent Michaelis Constant for BH\u003csub\u003e4\u003c/sub\u003e in the Cellular TPH System” for a detailed kinetic analysis. Statistical significance: two-way ANOVA with Dunnett's post-hoc test (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Error bars represent the mean ± SEM. The x-axis uses a logarithmic scale for the insets in (\u003cstrong\u003ec\u003c/strong\u003e) and (\u003cstrong\u003ed\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eThe numbered components in the TPH system (\u003cstrong\u003eb\u003c/strong\u003e) are (1) transporter, ENT; (2) sepiapterin reductase (SPR); (3) dihydrofolate reductase (DHFR); (4) tryptophan hydroxylase (TPH); (5) dihydropteridine reductase (DHPR); and (6) aromatic L-amino acid decarboxylase (AADC), which is inhibited by NSD-1015.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7300477/v1/0848d0a024136bfc7d738c02.png"},{"id":88751446,"identity":"4460ebe0-ff85-4b52-af5c-c7513704b191","added_by":"auto","created_at":"2025-08-11 06:22:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":183394,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSystemic administration of SP-enhanced brain TPH activity, accompanied by increased 5-HIAA levels.\u003c/strong\u003e \u003cstrong\u003e(a\u003c/strong\u003e) Underlying principle of TPH activity measurement using NSD-1015 metabolic arrest: Systemic application of NSD-1015 inhibits AADC and MAO, allowing 5HTP accumulation as a measure of TPH-mediated tryptophan conversion \u003cem\u003ein vivo\u003c/em\u003e. (\u003cstrong\u003eb\u003c/strong\u003e) Experimental design: C57BL/6j mice (\u003cem\u003en\u003c/em\u003e=6/group) were intraperitoneally administered SP (40 mg/kg) or vehicle, followed by NSD-1015 (100 mg/kg) or vehicle at 150 min. Brains were collected after 30 min of 5HTP accumulation at 180 min and divided into three regions: brainstem (A), cerebral cortex/hippocampus (B), and remaining forebrain (C). (\u003cstrong\u003ec–e\u003c/strong\u003e) Data are illustrated as box plots scaled by tissue weight (baseline: \u003cem\u003eblue\u003c/em\u003e, SP-treated: \u003cem\u003ered\u003c/em\u003e). (\u003cstrong\u003ec\u003c/strong\u003e) Regional TPH activity as nmol/g (measured as 30-min 5HTP accumulation per gram of tissue, +NSD conditions) showing heterogenous baseline activity (\u003cem\u003eblue\u003c/em\u003e) and enhanced activity by SP (\u003cem\u003ered\u003c/em\u003e). (\u003cstrong\u003ed\u003c/strong\u003e) Regional 5-HIAA levels (–NSD) demonstrating region-specific increases. (\u003cstrong\u003ee\u003c/strong\u003e) Regional 5-HIAA/5-HT ratios (–NSD) indicating enhanced serotonin turnover across all regions. Statistical significance vs. respective controls: *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001,\u003cem\u003e n.s.\u003c/em\u003e, not significant (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05) vs. respective controls.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7300477/v1/c2bf0590b15a785f6c0395c9.png"},{"id":88751137,"identity":"df127374-07c1-4ed1-ad7d-40a0ba60aab2","added_by":"auto","created_at":"2025-08-11 06:14:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":432885,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of systemic SP administration on brain BH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e, 5-HT, and 5-HIAA levels.\u003c/strong\u003e\u0026nbsp; (\u003cstrong\u003ea\u003c/strong\u003e) C57BL/6J mice received SP (0–125 mg/kg) intraperitoneally, and the brains were analyzed 6 h later for three regions: A (brainstem, \u003cem\u003ered\u003c/em\u003e), B (cortex/hippocampus, \u003cem\u003eblue\u003c/em\u003e), and C (remaining forebrain, \u003cem\u003egreen\u003c/em\u003e). (\u003cstrong\u003eb–d\u003c/strong\u003e) Panel numbering follows a matrix organization for clarity: raw data (row 1: \u003cstrong\u003eb\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e–d\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e), baseline values (row 2: \u003cstrong\u003eb\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e–d\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e), and normalized responses for regions A, B, and C (rows 3–5: suffix \u003cstrong\u003e3\u003c/strong\u003e, \u003cstrong\u003e4\u003c/strong\u003e, and \u003cstrong\u003e5\u003c/strong\u003e, respectively). Upper panels depict absolute amounts (nmol) with regions stacked; lower panels display fold-changes from baseline, wherein box dimensions represent concentration (height) × tissue weight (width). (\u003cstrong\u003eb\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e–b\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sub\u003e) BH\u003csub\u003e4\u003c/sub\u003e remained at baseline until approximately 20 mg/kg (threshold indicated by vertical lines), then increased linearly (\u003cem\u003edotted\u003c/em\u003e line, \u003cem\u003eR\u003c/em\u003e² as indicated) reaching 5.5-fold in Region A, 3.4-fold in Region B, and 3.0-fold in Region C at 125 mg/kg. (\u003cstrong\u003ec\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e–c\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sub\u003e) 5-HT displayed region-specific baseline differences (C \u0026gt; A \u0026gt; B) but minimal dose-dependency across all doses. (\u003cstrong\u003ed\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e–d\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sub\u003e) 5-HIAA exhibited threshold-dependent biphasic responses: below the approximate 20 mg/kg dose, Region A decreased to 82%, while B increased to 120% of baseline; above the threshold, all regions increased asymptotically, reaching 110%–150% at the maximum dose (B \u0026gt; A \u0026gt; C). Data are presented as mean ± SEM, n=7 (0–50 mg/kg), n=4 (125 mg/kg). Statistical significance: * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 versus baseline.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7300477/v1/8d10fcd6882ba86f87dc8103.png"},{"id":88751447,"identity":"b36993fc-2411-4780-9555-86ad97d20d77","added_by":"auto","created_at":"2025-08-11 06:22:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":465461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed schematic model of serotonin metabolic changes in response to systemic sepiapterin treatment\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eThis schematic model summarizes the key findings and restates the study objective of evaluating the modulation of brain serotonin metabolism by systemic sepiapterin (SP) via intracellular BH\u003csub\u003e4\u003c/sub\u003e elevation through targeted biochemical pathways. The model highlights: (1) a threshold dose (20 mg/kg) for 5-HIAA elevation; (2) region-specific serotonergic responses; (3) sustained BH\u003csub\u003e4\u003c/sub\u003e increase at 6 h post-administration; and (4) dose-proportional 5-HIAA increases beyond the threshold. These findings suggest that SP activates serotonin turnover by elevating BH\u003csub\u003e4\u003c/sub\u003e levels via salvage and recycling pathways. The diagram integrates these mechanisms to illustrate a unified model.\u003cbr\u003e\nPathway components are indicated as follows: [a] sepiapterin reductase, [b] dihydrofolate reductase, [c] tryptophan hydroxylase, [d] dihydropteridine reductase, [e] aromatic L-amino acid decarboxylase, [f] monoamine oxidase, [g] serotonin reuptake transporter, [h] BH\u003csub\u003e4\u003c/sub\u003e receptor (unknown), and [i/j] autoreceptors in brain regions A and B.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7300477/v1/a32c6b3b8fa9fed997b6be36.png"},{"id":88753109,"identity":"b5656067-7b4a-4ba7-b70e-bd7b29821ab7","added_by":"auto","created_at":"2025-08-11 06:46:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2481203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7300477/v1/b61dc1bb-aace-4988-a232-78905705ee3b.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eSepiapterin Enhances Brain Tetrahydrobiopterin BH4-Dependent Serotonin Synthesis with Regional Specificity\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSince the formulation of the serotonin hypothesis of depression in the 1960s (Coppen, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Ross \u003cem\u003eet al.\u003c/em\u003e, 1969), the enhancement of brain monoamine synthesis has remained a challenging therapeutic objective despite decades of extensive research. Tryptophan hydroxylase (TPH) and tyrosine hydroxylase (TH), the rate-limiting enzymes for serotonin and catecholamines, respectively, require tetrahydrobiopterin (BH\u003csub\u003e4\u003c/sub\u003e), the fully reduced form of biopterin, as a cofactor (Werner et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The activity of TPH adheres to Michaelis\u0026ndash;Menten kinetics regarding BH\u003csub\u003e4\u003c/sub\u003e concentration; however, its availability is associated with a recycling reaction mediated by dihydropteridine reductase (DHPR), which is crucial for monoamine synthesis.\u003c/p\u003e\u003cp\u003eBH\u003csub\u003e4\u003c/sub\u003e is synthesized \u003cem\u003ede novo\u003c/em\u003e from guanosine triphosphate (GTP), with the salvage pathway providing additional protection by converting the byproduct, sepiapterin (SP), to active BH\u003csub\u003e4\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This dual pathway system maintains intracellular BH\u003csub\u003e4\u003c/sub\u003e levels, which are essential for monoamine synthesis.\u003c/p\u003e\u003cp\u003eBH\u003csub\u003e4\u003c/sub\u003e deficiency severely impairs monoamine synthesis, resulting in profound neurological dysfunction. The strategy of supplementing TPH and TH with BH\u003csub\u003e4\u003c/sub\u003e to enable these enzymes to achieve their full potential was proposed several decades ago (Niederwieser et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1982\u003c/span\u003e), and peripheral 6R-BH\u003csub\u003e4\u003c/sub\u003e administration was adopted as the standard treatment (Opladen et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, the limited ability of 6R-BH4 to cross the blood\u0026ndash;brain barrier (BBB) has hindered its efficacy in enhancing central serotonin synthesis (Brand et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Recent studies have indicated that the administration of 6R-BH\u003csub\u003e4\u003c/sub\u003e stimulates the release of neurotransmitters, such as serotonin and dopamine, but with a minimal increase in transmitter synthesis (Fanet et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Winn et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This represents a major unmet medical need.\u003c/p\u003e\u003cp\u003eSepiapterin (SP) offers a potential solution as a membrane-permeable BH\u003csub\u003e4\u003c/sub\u003e precursor. Despite early \u003cem\u003ein vitro\u003c/em\u003e evidence of SP-enhanced TPH activity (Hasegawa et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), its translation to \u003cem\u003ein vivo\u003c/em\u003e brain applications was hindered by the prevailing view regarding rapid peripheral conversion and poor BBB penetration (Levine et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Our recent research demonstrates that SP above threshold doses crosses the BBB and enters brain cells through an ENT transporter coupled with the unidirectional BH\u003csub\u003e4\u003c/sub\u003e salvage pathway (Ohashi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) [\u003cb\u003epreprint\u003c/b\u003e]. Building on this foundation, the present study investigated the downstream effects of these changes on serotonin synthesis in the brain.\u003c/p\u003e\u003cp\u003eBased on this mechanistic insight, we systematically investigated SP-mediated enhancement of brain serotonin synthesis through: (1) \u003cem\u003ein vitro\u003c/em\u003e validation of SP-induced BH\u003csub\u003e4\u003c/sub\u003e elevation and TPH activation; (2) regional analysis of 5-HT and 5-HIAA in distinct brain compartments, assuming local metabolism without inter-regional trafficking; and (3) dose\u0026ndash;response characterization revealing biphasic effects, namely, subthreshold doses (\u0026lt;\u0026thinsp;20 mg/kg) allowed BH\u003csub\u003e4\u003c/sub\u003e to enter the brain without SP attainment and exhibited region-specific effects, whereas suprathreshold doses (\u0026gt;\u0026thinsp;20 mg/kg) produced robust increases in serotonin turnover because of SP reach. This design distinguishes the extracellular BH\u003csub\u003e4\u003c/sub\u003e effects from the intraneuronal SP-derived BH\u003csub\u003e4\u003c/sub\u003e function.\u003c/p\u003e\u003cp\u003eCollectively, our findings establish SP as an effective strategy for enhancing central serotonin biosynthesis through targeted TPH modulation. This strategy represents a mechanistically driven intervention targeting the rate-limiting step of 5-HT synthesis. This approach addresses the urgent need for BH\u003csub\u003e4\u003c/sub\u003e deficiency treatment and may benefit broader neuropsychiatric conditions involving impaired monoamine synthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThis study aimed to investigate three key hypotheses: (1) whether SP enters serotonergic neurons, (2) whether TPH activity is enhanced, and (3) whether serotonin synthesis increases above baseline. The question marks in the figure denote the key experimental targets.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cp\u003e2.1 Experimental Design Overview\u003c/p\u003e\n\u003cp\u003eWe used a two-phase approach: Initially, RBL2H3 cells were used to establish the mechanistic relationship between SP uptake, intracellular BH\u003csub\u003e4\u003c/sub\u003e elevation, and TPH activation. Subsequently, systemic SP administration in mice was used to investigate the dose-dependent effects on regional brain serotonin metabolism, focusing on TPH activity and serotonin turnover across anatomically distinct brain regions.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc203589862\"\u003e2.2 Materials\u003c/p\u003e\n\u003cp\u003eSepiapterin (SP) was obtained from Shiratori Pharmaceutical (Chiba, Japan). Tetrahydrobiopterin dihydrochloride (6R-BH\u003csub\u003e4\u003c/sub\u003e\u0026middot;2HCl) was obtained from Asubio Pharma (Kobe, Japan). 7,8-Dihydrobiopterin (BH\u003csub\u003e2\u003c/sub\u003e) was purchased from Schircks Laboratories (Jona, Switzerland), and N-Methylserotonin, a marker standard for high performance liquid chromatography (HPLC), and 3-hydroxybenzyl hydrazine (NSD-1015) were purchased from Nacalai Tesque (Kyoto, Japan).\u003c/p\u003e\n\u003cp id=\"_Toc203589863\"\u003e2.3 Cell-Culture Experiments\u003c/p\u003e\n\u003cp\u003eRBL2H3 cells (rat basophilic leukemia-derived serotonin-producing line; JCRB Cell Bank, Osaka, Japan) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with 10% fetal bovine serum at 37\u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. For the experiments, cells were seeded in 96-well plates (Falcon\u0026reg; 3072) at 10\u003csup\u003e5\u003c/sup\u003e cells/well and allowed to adhere overnight. To optimize TPH expression, cells were pretreated with A23187 (10 nM) for 2h, followed by 4 h incubation in \u0026quot;basal medium,\u0026rdquo; serum-free DMEM with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.2) and 1 mM dithiothreitol (DTT) (Hasegawa\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 1996).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBH\u003csub\u003e4\u003c/sub\u003e supplementation and TPH activity assessment:\u003c/em\u003e Reagents were refreshed by removing the previous medium, washing with Dulbecco\u0026rsquo;s phosphate buffered saline (DPBS), and replacing with a new basal medium containing the complete set of reagents. Cells were exposed to SP (0\u0026ndash;200 \u0026micro;M) or 6R-BH\u003csub\u003e4\u003c/sub\u003e (0\u0026ndash;1000 \u0026micro;M) for 30 min. To measure TPH activity, NSD-1015 (400 \u0026micro;M) was added for 30 min to inhibit\u0026nbsp;aromatic L-amino acid decarboxylase (AADC) and enable 5-hydroxytryptophan (5HTP) accumulation. Reactions were terminated by adding an equal volume of 0.8 M perchloric acid containing 2 mM DTT, and the cells and medium were harvested. For SP/6R-BH\u003csub\u003e4\u003c/sub\u003e uptake measurement, basal medium without NSD-1015 was added for 30 min, followed by treatment of cells with acidic or alkaline I\u003csub\u003e2\u003c/sub\u003e solutions, as described in \u003cem\u003esection\u003c/em\u003e \u003cstrong\u003e2.5\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp id=\"_Toc203589864\"\u003e2.4 Mouse Experiments\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by Nihon University Animal Experiment Committee (AP13M037) and complied with the ARRIVE Guidelines 2.0. Six-week-old male C57BL/6J mice (CLEA Japan,\u0026nbsp;Inc., Tokyo, Japan) were housed under standard conditions (12\u003cstrong\u003e-\u003c/strong\u003eh light/dark cycle, 21\u003cstrong\u003e\u0026ndash;\u003c/strong\u003e24\u0026deg;C, 40%\u0026ndash;60% humidity) with \u003cem\u003ead libitum\u003c/em\u003e access to the experimental diet MF (Oriental Yeast, Tokyo,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eJapan) and water. Experiments were conducted between 10:00 and 17:00 (Japan Standard Time; JST) after a one-week acclimation period. At the conclusion of the experiment, each mouse was sacrificed by decapitation for brain collection.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAssessment of TPH activity\u003c/em\u003e (\u003cem\u003eFig.\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e): Mice (mean weight: 21.7 \u0026plusmn; 1.2 g, n=6/group) were administered SP in saline (2 mg/mL) at a dosage of 40 mg/kg or intraperitoneal saline at t = 0, followed by NSD-1015 (5 mg/mL, 100 mg/kg) or PBS(\u0026ndash;) administered intraperitoneally at t = 150 min. Brains were collected at t = 180 min.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDose\u0026ndash;response study\u003c/em\u003e (\u003cem\u003eFig.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e): Mice (mean weight: 23.9 \u0026plusmn; 0.9 g) were administered with SP in saline (2 mg/kg) at doses of 0, 10, 20, 50 mg/kg (n=7/dose) or 125 mg/kg (n=4) intraperitoneally. Brains were harvested 6 h post-administration, when intracellular BH\u003csub\u003e4\u003c/sub\u003e could be selectively quantified because of the clearance of extracellular BH\u003csub\u003e4\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBrain dissection:\u003c/em\u003e A functional division approach was used to differentiate serotonergic cell bodies from projection territories (detailed protocol in\u003cstrong\u003e\u0026nbsp;S1. Supplementary Methods\u003c/strong\u003e). Briefly, the brains were dissected into three regions: Region A (brainstem with raphe nuclei), Region B (cortex/hippocampus), and Region C (subcortical structures). The coefficients of variation (CVs; mean weight \u0026plusmn; SD mg, n = 25) were as follows: Region A, 0.062 (47.1 \u0026plusmn; 2.9); Region B, 0.033 (242 \u0026plusmn; 8); and Region C, 0.065 (90.3 \u0026plusmn; 5.9). This approach enables a quantitative assessment of the regional serotonin synthesis capacity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTissue processing:\u0026nbsp;\u003c/em\u003eFrozen tissues were homogenized using a mechanical homogenizer (T 25-S1, IKA Labortechnik, Breisgau, Germany) on ice in 0.1 M HCl containing 0.5 \u0026micro;M N-methylserotonin (internal standard) and aliquoted for biochemical analyses.\u003c/p\u003e\n\u003cp id=\"_Toc203589865\"\u003e2.5 Biochemical Determinations\u003c/p\u003e\n\u003cp\u003eBiopterin measurements:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe Fukushima\u0026ndash;Nixon differential oxidation method was used for biopterin quantification (Fukushima\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 1980). This technique provides stable and reproducible results for large sample sizes. In this method, acidic I\u003csub\u003e2\u003c/sub\u003e oxidation yields total reduced biopterins, BH\u003csub\u003e4\u003c/sub\u003e, biopterin-4a-carbinilamine, quinonoid dihydrobiopterin (qBH\u003csub\u003e2\u003c/sub\u003e), and BH\u003csub\u003e2\u003c/sub\u003e, whereas alkaline I\u003csub\u003e2\u003c/sub\u003e oxidation yields only BH\u003csub\u003e2\u003c/sub\u003e. In this manuscript, \u0026quot;BH\u003csub\u003e4\u003c/sub\u003e\u0026quot; represents physiologically relevant cofactor levels, although measurements represent total reduced biopterins (tBH\u003csub\u003e4\u003c/sub\u003e) from acid oxidation alone, thereby avoiding calculation errors from subtraction.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSample preparation:\u003c/em\u003e For cultured cells (\u003cem\u003eFig\u003c/em\u003e. \u003cstrong\u003e2\u003c/strong\u003e), the cells in the wells were washed with DPBS and directly treated with 50 \u0026micro;L iodine solution. For brain tissue (\u003cem\u003eFig\u003c/em\u003e. \u003cstrong\u003e4\u003c/strong\u003e), frozen homogenates (50 \u0026micro;L aliquots in 0.1 M HCl) were thawed on ice and mixed with an equal volume of iodine solution. All procedures were performed under dim light to avoid photodegradation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eOxidation procedure:\u003c/em\u003e Samples were treated with acidic I₂ (2% I₂, 3% KI in 0.1 M HCl) or alkaline I₂ (2% I₂, 3% KI in 0.2 M NaOH), incubated at 30\u0026deg;C for 60 min, and then quenched with an equal volume of 0.2 M ascorbic acid in 1 M perchloric acid. After centrifugation (9,100 \u0026times; g, 10 min, 4\u0026deg;C), the supernatants were analyzed using HPLC (octadecylsilica (ODS) column: JASCO Finepak SIL C18T-5, 40˚C, 7% methanol, fluorescence detection: \u003cem\u003eE\u003c/em\u003ex = 350, \u003cem\u003eE\u003c/em\u003em = 450 nm). A mixture of authentic biopterin and 2-amino-4-hydroxypteridine (pterin) was run as an external standard every 10 analyses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRationale for tBH\u003csub\u003e4\u003c/sub\u003e reporting:\u003c/em\u003e In the TPH-DHPR coupled system, BH\u003csub\u003e4\u003c/sub\u003e and qBH\u003csub\u003e2\u003c/sub\u003e were dynamically partitioned between enzymes. DHFR in the BH\u003csub\u003e4\u003c/sub\u003e salvage pathway rescues most of the BH\u003csub\u003e2\u003c/sub\u003e. Reporting acid oxidation values (tBH\u003csub\u003e4\u003c/sub\u003e) reflects the physiologically relevant cofactor pool without introducing any subtraction errors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5HTP, 5-HT, and 5-HIAA:\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe indole compounds were quantified using HPLC with native fluorescence detection as previously described (Hasegawa\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 1987), with minor modifications specific to 5HTP measurement in brain tissue. Culture cells or tissue homogenates were mixed with perchloric acid/DTT solution (final concentrations: 0.4 M and 1 mM). After centrifugation (9,100 \u0026times; \u003cem\u003eg\u003c/em\u003e, 10 min, 4 \u0026deg;C), the supernatant was analyzed using an ODS column (JASCO Finepak SIL C18T-5, 40 \u0026deg;C) with fluorescence detection (\u003cem\u003eE\u003c/em\u003ex: 302 nm, \u003cem\u003eE\u003c/em\u003em: 350 nm). The mobile phase consisted of 10 mM sodium acetate (pH 3.5, adjusted with formic acid), acetonitrile, and methanol (100:5:7, v/v/v). External standards containing tryptophan, 5-HTP, 5-HT, 5-HIAA, and N-methylserotonin were run after every 10 samples. Specifically, for the \u003cem\u003ein situ\u003c/em\u003e TPH activity assay (\u003cem\u003eFig.\u003c/em\u003e \u003cstrong\u003e3c\u003c/strong\u003e), the mobile phase was modified to a 100:9:1.5 mixture to separate 5HTP from brain-specific contaminants.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.6 Statistical Analysis\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; SEM with individual data points. Statistical analyses were performed using Excel with the Bell Curve add-in (Social Research Information; Tokyo, Japan). Two-way analysis of variance (ANOVA) with Dunnett\u0026apos;s test was used for the \u003cem\u003ein vitro\u003c/em\u003e dose\u0026ndash;response analysis (\u003cem\u003eFig.\u003c/em\u003e \u003cstrong\u003e2d\u003c/strong\u003e). Multi-way ANOVA with Tukey\u0026apos;s post-hoc comparisons was applied to the \u003cem\u003ein vivo\u003c/em\u003e experiments (\u003cem\u003eFigs.\u003c/em\u003e \u003cstrong\u003e3\u0026ndash;4\u003c/strong\u003e). For the kinetic analysis in\u0026nbsp;\u003cstrong\u003e\u003cem\u003eFig.\u003c/em\u003e2\u003c/strong\u003e,\u0026nbsp;TPH activity data were fitted to both simple (\u003cem\u003en\u003c/em\u003e = 1) and extended (\u003cem\u003en\u003c/em\u003e \u0026gt; 1) Michaelis\u0026ndash;Menten equations using non-linear regression. The extended equation is\u003cbr\u003e\u003cem\u003ev\u003c/em\u003e = \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e\u0026middot;[\u003cem\u003eS\u003c/em\u003e]\u003cem\u003eⁿ\u003c/em\u003e/((app\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e)\u003cem\u003eⁿ\u003c/em\u003e + [\u003cem\u003eS\u003c/em\u003e]\u003cem\u003eⁿ\u003c/em\u003e)\u003cbr\u003ewhere \u003cem\u003ev\u003c/em\u003e is the reaction velocity, [\u003cem\u003eS\u003c/em\u003e] is the substrate concentration (tBH\u003csub\u003e4\u003c/sub\u003e), \u003cem\u003en\u003c/em\u003e is the Hill coefficient, and app\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e is the apparent Michaelis constant.\u0026nbsp;For \u003cstrong\u003e\u003cem\u003eFig.\u003c/em\u003e\u003c/strong\u003e \u003cstrong\u003e4\u003c/strong\u003e, raw data analyses (\u003cem\u003eupper\u003c/em\u003e panels) determined statistical significance, with effect sizes calculated using Hedges\u0026apos; \u003cem\u003eg\u003c/em\u003e.\u0026nbsp;Normalized data (\u003cem\u003elower\u003c/em\u003e panels) show the same datasets as fold-changes, preserving statistical significance while facilitating the interpretation of relative changes across regions. Linear regression was used to determine the threshold doses from the x-intercepts.\u0026nbsp;Effect size interpretations followed Cohen\u0026apos;s conventions: \u003cem\u003e\u0026eta;\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (0.01 = small, 0.06 = medium, 0.14 = large) and Hedges\u0026apos; \u003cem\u003eg\u003c/em\u003e (0.2 = small, 0.5 = medium, 0.8 = large).\u0026nbsp;Post-hoc power analysis was conducted for the dose\u0026ndash;response study. For the 125 mg/kg group (n=4), with the observed effect size (Cohen\u0026apos;s \u003cem\u003ed\u003c/em\u003e = 1.33), the achieved power was 0.47. Sample size calculations indicated that n=9 per group would achieve 80% power for detecting similar effect sizes.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Cellular Uptake of SP/6R-BH\u003csub\u003e4\u003c/sub\u003e and Their Effects on TPH Activity\u003c/h2\u003e\n \u003cp\u003eThe mechanistic relationship between extracellular SP/6R-BH\u003csub\u003e4\u003c/sub\u003e exposure and intracellular TPH activation was examined using serotonin-producing RBL2H3 cells. Cells were exposed to varying concentrations of SP (0\u0026ndash;200 \u0026micro;M) or 6R-BH\u003csub\u003e4\u003c/sub\u003e (0\u0026ndash;1000 \u0026micro;M) for 30 min, followed by the measurement of intracellular BH\u003csub\u003e4\u003c/sub\u003e levels (tBH\u003csub\u003e4\u003c/sub\u003e) and TPH activity (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). SP demonstrated markedly superior cellular uptake compared to that of 6R-BH\u003csub\u003e4\u003c/sub\u003e. Linear regression analysis revealed uptake rates of 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 pmol/well per \u0026micro;M for SP (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.993, n\u0026thinsp;=\u0026thinsp;32) vs. 0.031\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 pmol/well per \u0026micro;M for 6R-BH\u003csub\u003e4\u003c/sub\u003e (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.937, n\u0026thinsp;=\u0026thinsp;48), representing a 21-fold difference. This differential uptake efficiency is attributed to the substrate specificity of the ENT2 transporter and the subsequent conversion of SP to BH\u003csub\u003e4\u003c/sub\u003e via the salvage pathway (Ohashi et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sawabe et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). Enhancement of TPH activity exhibited distinct concentration-response patterns: SP achieved maximum activation at 30\u0026ndash;50 \u0026micro;M extracellular concentration, whereas 6R-BH\u003csub\u003e4\u003c/sub\u003e required\u0026thinsp;\u0026gt;\u0026thinsp;500 \u0026micro;M for comparable effects. To achieve a 25% increase in TPH activity over the baseline, approximately 0.1 \u0026micro;M SP or 3 \u0026micro;M 6R-BH\u003csub\u003e4\u003c/sub\u003e was required. Notably, when TPH activity was plotted against the achieved intracellular BH\u003csub\u003e4\u003c/sub\u003e levels rather than extracellular exposure concentrations, the data from both treatments converged onto a single curve (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). This convergence indicates that TPH activation is solely dependent on intracellular BH\u003csub\u003e4\u003c/sub\u003e levels, with enhanced cellular uptake being entirely responsible for the superior efficacy of SP.\u003c/p\u003e\n \u003cp\u003eThe cellular TPH system operates as a coupled enzyme complex, wherein TPH activity is intricately associated with BH\u003csub\u003e4\u003c/sub\u003e recycling via DHPR. An analysis of the TPH reaction velocity in relation to total intracellular total BH\u003csub\u003e4\u003c/sub\u003e (tBH\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;BH\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;qBH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;biopterin-4a-carbinolamine) revealed cooperative kinetics fitting the extended Michaelis\u0026ndash;Menten equation:\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:v=\\frac{{V}_{\\text{m}\\text{a}\\text{x}}\\cdot\\:{\\left[S\\right]}^{n}}{{{\\text{a}\\text{p}\\text{p}K}_{\\text{M}}}^{n}+{\\left[S\\right]}^{n}}\\)\u003c/span\u003e\u003c/span\u003e \u0026hellip;\u0026hellip; (1).\u003c/p\u003e\n \u003cp\u003eNon-linear regression yielded \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.1, \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e = 40.0 pmol/30-min/well, and app\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e = 18.1 \u0026micro;M (\u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.906, n\u0026thinsp;=\u0026thinsp;81). The extended model provided a significantly better fit compared to simple Michaelis\u0026ndash;Menten kinetics (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1; \u003cem\u003eR\u003c/em\u003e\u0026sup2; = 0.848), confirming cooperative substrate binding. The Hill coefficient of 2.1 suggests positive cooperativity within the TPH-DHPR complex. Notably, app\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e approximated the endogenous tBH\u003csub\u003e4\u003c/sub\u003e concentration (17.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026micro;M, estimated using standard assumptions of 1.80 pL spherical cell volume and 1\u0026times;10⁵ cells/well, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), suggesting that cells maintain BH\u003csub\u003e4\u003c/sub\u003e levels near the \u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e for optimal responsiveness to cofactor fluctuations. This coupling creates a fundamental constraint: since \u003cem\u003ev\u003c/em\u003e\u003csub\u003eTPH\u003c/sub\u003e = \u003cem\u003ev\u003c/em\u003e\u003csub\u003eDHPR\u003c/sub\u003e in the recycling system, and [BH\u003csub\u003e4\u003c/sub\u003e] + [qBH\u003csub\u003e2\u003c/sub\u003e] = [tBH\u003csub\u003e4\u003c/sub\u003e]. Hence, the available BH\u003csub\u003e4\u003c/sub\u003e for TPH is always less than the total tBH\u003csub\u003e4\u003c/sub\u003e. As such, DHPR both sustains and limits TPH activity, explaining why, even in the presence of high BH\u003csub\u003e4\u003c/sub\u003e concentrations, cellular systems cannot reach the theoretical \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e of either enzyme.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 TPH activation \u003cem\u003ein vivo\u003c/em\u003e is accompanied by increased 5-HIAA across brain regions\u003c/h2\u003e\n \u003cp\u003eTo establish a region-specific analysis of SP effects on serotonin metabolism, TPH activity was assessed 2.5 h following intraperitoneal SP administration (40 mg/kg) using metabolic arrest with NSD-1015 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The brains were divided into three regions based on serotonergic anatomy: brainstem containing cell bodies (Region A, 47.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 mg), cortex/hippocampus with projections (Region B, 242\u0026thinsp;\u0026plusmn;\u0026thinsp;8 mg), and the remaining forebrain (Region C, 90.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.9 mg). Baseline TPH activity exhibited pronounced regional heterogeneity (ANOVA: \u003cem\u003eF\u003c/em\u003e (2,22)\u0026thinsp;=\u0026thinsp;285, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with Regions B and C exhibiting approximately twice the activity of Region A (ratio 1:1.81:1.94), reflecting axonal TPH enzyme transport from the brainstem cell bodies. SP administration enhanced TPH activity differentially across regions (\u003cem\u003eF\u003c/em\u003e (1,11)\u0026thinsp;=\u0026thinsp;6.23, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030), with significant increases in Regions A (+\u0026thinsp;12%) and C (+\u0026thinsp;16%, both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). SP-induced distinct patterns of 5-HIAA changes: Region B exhibited the most pronounced increase (+\u0026thinsp;67%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while 5-HIAA/5-HT ratios increased significantly across all regions (A: +23%, B: +67%, C: +31%; all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). These differential responses demonstrate that (1) brain regions must be analyzed separately because of distinct TPH distribution patterns and (2) concurrent 5-HIAA measurement is essential for evaluating serotonin turnover. This single-dose study established a methodological framework for a comprehensive dose\u0026ndash;response analysis required to elucidate the region-specific effects of SP on serotonin metabolism. Subsequently, we conducted comprehensive dose\u0026ndash;response studies across multiple brain regions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Dose-Dependent Effects of SP on Brain BH\u003csub\u003e4\u003c/sub\u003e and Serotonin Metabolism\u003c/h2\u003e\n \u003cp\u003eSystemic SP administration (0\u0026ndash;125 mg/kg) intraperitoneally induced dose-dependent changes in brain BH\u003csub\u003e4\u003c/sub\u003e and monoamine metabolism at 6 h post-injection (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, organized in matrix format for systematic regional comparisons), when intracellular BH\u003csub\u003e4\u003c/sub\u003e can be selectively quantified owing to the clearance of extracellular BH\u003csub\u003e4\u003c/sub\u003e (Ohashi et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) [\u003cstrong\u003epreprint\u003c/strong\u003e]. To evaluate these effects across brain regions with different baseline concentrations, we analyzed both the absolute amounts (\u003cem\u003eupper\u003c/em\u003e panels) and fold-changes from the baseline (\u003cem\u003elower\u003c/em\u003e panels). Absolute measurements revealed dose-dependent increases in total brain BH\u003csub\u003e4\u003c/sub\u003e (\u003cem\u003eFig.\u0026nbsp;4\u003c/em\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e), reaching a 3.4-fold elevation at 125 mg/kg (ANOVA: \u003cem\u003eF\u003c/em\u003e(4,26)\u0026thinsp;=\u0026thinsp;15.3, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). 5-HT levels showed modest variations (\u003cem\u003eFig.\u0026nbsp;4\u003c/em\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e; \u003cem\u003eF\u003c/em\u003e(4,26)\u0026thinsp;=\u0026thinsp;1.69, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.184), whereas 5-HIAA exhibited significant changes (\u003cem\u003eFig.\u0026nbsp;4\u003c/em\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e1\u003c/strong\u003e\u003c/sub\u003e; \u003cem\u003eF\u003c/em\u003e(4,26)\u0026thinsp;=\u0026thinsp;6.97, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003e\u0026eta;\u003c/em\u003e\u0026sup2; = 0.517, very large effect). Given the substantial differences in baseline concentrations across regions (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cem\u003eleftmost\u003c/em\u003e columns), we normalized the data to the baseline values. This normalization revealed distinct responses, suggesting that the threshold effect of the SP dose was masked by baseline differences. This convergent threshold suggests a systemic barrier to BH\u003csub\u003e4\u003c/sub\u003e elevation at lower doses. The dose-dependent changes in effect sizes, from negative values at 10\u0026ndash;20 mg/kg (Hedges\u0026apos; \u003cem\u003eg\u003c/em\u003e = \u0026minus;\u0026thinsp;0.58 to \u0026minus;\u0026thinsp;0.28) to moderate positive effects at 50 mg/kg (\u003cem\u003eg\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.442) and large effects at 125 mg/kg (\u003cem\u003eg\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.758), demonstrate a clear threshold phenomenon. Despite moderate statistical power at 125 mg/kg (1\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003e\u0026beta;\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.47, n\u0026thinsp;=\u0026thinsp;4), the large effect size confirms robust biological activity above the threshold dose, consistent with the ~\u0026thinsp;20 mg/kg threshold determined by linear regression and BH₄-mediated TPH activation.\u003c/p\u003e\n \u003cp\u003eBH\u003csub\u003e4\u003c/sub\u003e responses: Brain BH\u003csub\u003e4\u003c/sub\u003e exhibited a biphasic dose-dependency (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). No significant increases were observed below 20 mg/kg in any of the regions. Normalized BH\u003csub\u003e4\u003c/sub\u003e responses displayed remarkable linearity above the threshold SP dose, which was consistent across regions A, B, and C, with regression lines intercepting the x-axis at 17.8 (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.69), 11.7 (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.77), and 11.45 (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.81) mg/kg, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e\u0026ndash;b\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sub\u003e, dashed lines). The baseline BH\u003csub\u003e4\u003c/sub\u003e abundance in Region B was 3-fold higher than that in Region A (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e), consistent with the 1.81-fold higher TPH activity (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e5-HT levels\u003c/em\u003e: Regional analysis revealed heterogeneous responses (multi-way ANOVA: \u003cem\u003eF\u003c/em\u003e(2,52)\u0026thinsp;=\u0026thinsp;5.49, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007). Region A exhibited biphasic changes, with significant decreases at 10\u0026ndash;50 mg/kg, followed by an elevation at 125 mg/kg (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Region B maintained stable 5-HT levels throughout, whereas Region C displayed modest elevation trends at high doses.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e5-HIAA patterns\u003c/em\u003e: Two distinct dose-dependent patterns emerged, demarcated by the BH\u003csub\u003e4\u003c/sub\u003e threshold (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). Below the threshold, contrasting regional responses occurred: Region A showed decreased 5-HIAA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049), while Region B showed an increase (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025), with no net whole-brain change because of Region B\u0026apos;s larger tissue mass. Above the threshold, all regions exhibited dose-dependent 5-HIAA increases (Region A: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Region B: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Region C: \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), accompanied by maintained or increased 5-HT levels, thereby demonstrating a successful enhancement of 5-HT synthesis. These biphasic 5-HIAA responses indicate two distinct mechanisms: below the threshold, peripherally-generated BH\u003csub\u003e4\u003c/sub\u003e induces region-specific monoamine release without enhancing its synthesis. Above this threshold, SP-derived intracellular BH\u003csub\u003e4\u003c/sub\u003e enhances TPH activity, increasing both synthesis and turnover. This dual mechanism explains the complex dose\u0026ndash;response relationships and establishes the optimal dosing requirements for therapeutic serotonin enhancement.\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study revealed that peripheral sepiapterin (SP) enhances brain serotonin synthesis through a defined route: crossing the BBB, entering neurons via transporters, increasing BH\u003csub\u003e4\u003c/sub\u003e levels, and activating TPH. Rather than data-driven screening, this is a hypothesis-driven intervention targeting a known biosynthetic bottleneck. Our results revealed clear region- and dose-dependent associations between SP and BH\u003csub\u003e4\u003c/sub\u003e elevation and the serotonin turnover. A summary of the model is presented in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1 Mechanistic Interpretation at the Cellular Level\u003c/h2\u003e\n \u003cp\u003eOur cellular studies revealed that SP represents a superior strategy for enhancing brain serotonin synthesis compared to direct BH\u003csub\u003e4\u003c/sub\u003e supplementation. Using intact RBL2H3 cells, which share key functional properties with serotonergic neurons that project throughout the brain, including serotonin production, storage, and release, we preserved cellular integrity to clarify the critical role of precursor transport in TPH enhancement.\u003c/p\u003e\n \u003cp\u003eThe observed superior uptake efficiency of SP compared to that of 6R-BH\u003csub\u003e4\u003c/sub\u003e reflects the substrate specificity of ENT2 (6R-BH\u003csub\u003e4\u003c/sub\u003e:BH\u003csub\u003e2\u003c/sub\u003e:SP ratio\u0026thinsp;=\u0026thinsp;1:1.92:15.6) and the synergistic effect of the push\u0026ndash;pull drive of SP through the salvage pathway (Sawabe et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). The key transporter, ENT2, is abundant throughout the brain parenchyma (Lu et al., \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e). This differential transport efficiency translates directly to enhanced TPH activation (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e): achieving equivalent intracellular BH\u003csub\u003e4\u003c/sub\u003e levels requires impractically higher 6R-BH\u003csub\u003e4\u003c/sub\u003e exposure. The convergence of SP and 6R-BH\u003csub\u003e4\u003c/sub\u003e data when plotted against intracellular BH\u003csub\u003e4\u003c/sub\u003e concentrations confirms that precursors function equivalently as coenzymes once internalized, with efficacy differences explained solely by uptake efficiency, which in turn confirms that extracellular BH\u003csub\u003e4\u003c/sub\u003e remains extracellular and is not efficiently taken up into the cells.\u003c/p\u003e\n \u003cp\u003eThe obligate coupling between TPH and DHPR activities creates unique kinetic constraints within the cellular system. Our analysis revealed cooperative kinetics (Hill coefficient\u0026thinsp;=\u0026thinsp;2.1) with an app\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e of 18.1 \u0026micro;M, which was remarkably close to the endogenous tBH\u003csub\u003e4\u003c/sub\u003e levels (17.9 \u0026micro;M). This alignment suggests that cells maintain BH\u003csub\u003e4\u003c/sub\u003e near the \u0026quot;\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e level\u0026quot; for optimal responsiveness, consistent with previous findings that intracerebroventricular 6R-BH\u003csub\u003e4\u003c/sub\u003e can double brain TPH activity, indicating subsaturating baseline BH\u003csub\u003e4\u003c/sub\u003e levels (Miwa et al., \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eBH\u003csub\u003e2\u003c/sub\u003e, which is derailed from the BH\u003csub\u003e4\u003c/sub\u003e redox cycle by spontaneous tautomerization (qBH\u003csub\u003e2\u003c/sub\u003e\u0026rarr;BH\u003csub\u003e2\u003c/sub\u003e, \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e = 1.6 min (Watabe, \u003cspan class=\"CitationRef\"\u003e1978\u003c/span\u003e)), is not recycled by DHPR. Instead, it is primarily recovered via the salvage pathway, and the remaining portion leaks out of the cell (permeability BH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;BH\u003csub\u003e4\u003c/sub\u003e), resulting in substantial BH\u003csub\u003e4\u003c/sub\u003e loss. This loss of cellular BH\u003csub\u003e4\u003c/sub\u003e is compensated by \u003cem\u003ede novo\u003c/em\u003e synthesis. Critically, the recycling of BH\u003csub\u003e4\u003c/sub\u003e between TPH and DHPR ensures a stable supply of BH\u003csub\u003e4\u003c/sub\u003e, but limits the available BH\u003csub\u003e4\u003c/sub\u003e to less than the total tBH\u003csub\u003e4\u003c/sub\u003e. A weak DHPR increases qBH2 in the equation [BH\u003csub\u003e4\u003c/sub\u003e] = [tBH\u003csub\u003e4\u003c/sub\u003e] - [qBH\u003csub\u003e2\u003c/sub\u003e]. Regardless of the concentration of tBH\u003csub\u003e4\u003c/sub\u003e, the resulting TPH activity will be lower than its potential activity, that is, app\u003cem\u003eV\u003c/em\u003e\u003csub\u003emax\u003c/sub\u003e \u0026lt; \u003cem\u003eV\u003c/em\u003e\u003csub\u003emax,TPH\u003c/sub\u003e. This explains why DHPR deficiency impairs monoamine production despite significant BH\u003csub\u003e4\u003c/sub\u003e levels being maintained (Xu et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), which is a key insight for understanding inherited metabolic disorders. Although specific regulatory details may differ between RBL2H3 cells and authentic serotonergic neurons, these fundamental characteristics of BH\u003csub\u003e4\u003c/sub\u003e-dependent hydroxylase systems are likely to be broadly applicable.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e4.2 Regional Characteristics of the Serotonergic System at Baseline\u003c/h2\u003e\n \u003cp\u003eRegional analysis has provided fundamental insights into the anatomical and functional organization of serotonin production in the brain. Previous studies have shown that GTPCH1 is primarily expressed in serotonergic neurons, with a lesser but significant contribution from catecholaminergic neurons (Hirayama \u003cem\u003eet al.\u003c/em\u003e, 1998; Lentz \u003cem\u003eet al.\u003c/em\u003e, 1996). The baseline tBH\u003csub\u003e4\u003c/sub\u003e distribution shown in \u003cstrong\u003eFig.\u0026nbsp;4\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e aligned with the GTPCH1 expression pattern. It is highest in Region A containing serotonergic cell bodies, intermediate in Region C with mixed serotonergic projections and dopaminergic neurons, and lowest concentration in Region B.\u003c/p\u003e\n \u003cp\u003eAlthough the tBH\u003csub\u003e4\u003c/sub\u003e concentration in Region B is approximately half that in Region A, its tBH\u003csub\u003e4\u003c/sub\u003e content (nmol/g \u0026times; g) is three times greater because of the larger tissue volume. Based on the empirical consistency of intracellular BH\u003csub\u003e4\u003c/sub\u003e being close to app\u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e (approximately 18 \u0026micro;M\u0026thinsp;\u0026asymp;\u0026thinsp;1.8 nmol/g), the tBH\u003csub\u003e4\u003c/sub\u003e concentration in Region A (0.82 nmol/g) corresponds to 2.1 mg of cytoplasm containing 1.8 nmol/g tBH\u003csub\u003e4\u003c/sub\u003e, which represents 4.4% of the tissue mass (47.1 mg). Presumably, this represents the cytoplasmic volume of serotonergic neurons in the brainstem. Similarly, 6.52 mg of cytoplasm maintained TPH activity in Region B, corresponding to 2.7% of the tissue mass (242 mg).\u003c/p\u003e\n \u003cp\u003eThe baseline activity of TPH is maintained by the supply of consumed BH\u003csub\u003e4\u003c/sub\u003e via DHPR recycling. Specifically, the TPH activity per tBH\u003csub\u003e4\u003c/sub\u003e concentration, that is, the cofactor utilization rate, was lower in region B than in Region A (compare Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec with \u003cem\u003eFig.\u0026nbsp;4\u003c/em\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e). As efficient cofactor utilization is determined by the DHPR/TPH ratio via the partitioning of tBH\u003csub\u003e4\u003c/sub\u003e to BH\u003csub\u003e4\u003c/sub\u003e and qBH\u003csub\u003e2\u003c/sub\u003e, it is unclear whether this regional difference is because of TPH or DHPR insufficiency. This quantitative demonstration that axonal varicosities and synaptic terminals in projection regions (Region B) actively produce serotonin challenges the traditional view of limiting synthesis to cell body regions. These baseline characteristics establish the foundation for understanding the differential regional effects of SP and highlight the importance of analyzing both synthesis capacity and metabolic turnover across anatomically distinct serotonergic compartments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e4.3 Biphasic Response Mechanisms\u003c/h2\u003e\n \u003cp\u003eThe threshold phenomenon at ~\u0026thinsp;20 mg/kg SP represents a pivotal discovery that explains the previously inconsistent findings regarding SP efficacy. This threshold reflects the competition between peripheral and central SP uptake, with peripheral organs preferentially sequestering SP at low doses.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eLow-dose mechanism (\u0026lt;\u0026thinsp;20 mg/kg)\u003c/em\u003e: Below this threshold, SP is rapidly converted to BH\u003csub\u003e4\u003c/sub\u003e in peripheral organs, with subsequent systemic BH\u003csub\u003e4\u003c/sub\u003e delivery to the brain, but is largely cleared after 6 h (Ohashi et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) [\u003cstrong\u003epreprint\u003c/strong\u003e]. This peripherally-derived BH\u003csub\u003e4\u003c/sub\u003e, while entering the brain parenchyma, remains predominantly extracellular and triggers monoamine release without enhancing monoamine synthesis\u0026mdash;consistent with the findings of previous 6R-BH\u003csub\u003e4\u003c/sub\u003e studies showing release stimulation independent of synthesis promotion (Fanet et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e; Koshimura et al., \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e; Winn et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). The contrasting regional 5-HIAA responses (decreased in Region A increased in Region B) likely reflect the differential expression of serotonin autoreceptors: somatodendritic 5-HT1A receptors in Region A providing inhibitory feedback versus terminal 5-HT1B receptors in Region B modulating release dynamics. This explains why cellular studies show linear SP responses (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e), whereas \u003cem\u003ein vivo\u003c/em\u003e responses exhibit threshold behavior.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eHigh-dose mechanism (\u0026gt;\u0026thinsp;20 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAbove this threshold, peripheral SP uptake saturates, allowing systemic SP circulation and brain delivery. SP crosses the BBB, enters neurons via ENT2 transporters, and is converted to intracellular BH\u003csub\u003e4\u003c/sub\u003e via the salvage pathway. This intracellular BH\u003csub\u003e4\u003c/sub\u003e enhancement follows kinetics similar to those of cultured cells (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee), with TPH activity increasing according to cellular BH\u003csub\u003e4\u003c/sub\u003e levels. Notably, the regional heterogeneity observed at low doses was reversed at high doses, producing uniform serotonin parameter increases across all regions. This suggests that enhanced intracellular BH\u003csub\u003e4\u003c/sub\u003e-driven synthesis overwhelms the region-specific release modulation observed at low doses, providing a unified mechanism for therapeutic serotonin enhancement in diverse brain regions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003e4.4 Comparison with Existing Research and Mechanistic Implications\u003c/h2\u003e\n \u003cp\u003eOur discovery of the biphasic dose\u0026ndash;response pattern of SP provides a mechanistic framework for addressing four decades of challenges in BH\u003csub\u003e4\u003c/sub\u003e-based therapeutics. Since Niederwieser et al.\u0026rsquo;s seminal work in the 1980s (Curtius et al., \u003cspan class=\"CitationRef\"\u003e1983\u003c/span\u003e; Niederwieser et al., \u003cspan class=\"CitationRef\"\u003e1982\u003c/span\u003e), 6R-BH\u003csub\u003e4\u003c/sub\u003e has been the standard for BH\u003csub\u003e4\u003c/sub\u003e replacement, despite demonstrating inferior cellular uptake compared to SP (Hasegawa et al., \u003cspan class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sawabe et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). The persistent use of 6R-BH\u003csub\u003e4\u003c/sub\u003e in the field has overlooked a critical distinction: while 6R-BH\u003csub\u003e4\u003c/sub\u003e increases brain BH\u003csub\u003e4\u003c/sub\u003e levels, this BH\u003csub\u003e4\u003c/sub\u003e remains predominantly extracellular, stimulating monoamine release without enhancing its synthesis (Koshimura et al., \u003cspan class=\"CitationRef\"\u003e1994\u003c/span\u003e). This explains why congenital BH\u003csub\u003e4\u003c/sub\u003e deficiency syndromes require combination therapy with L-DOPA and 5HTP, rather than 6R-BH\u003csub\u003e4\u003c/sub\u003e monotherapy (Opladen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, to avoid adverse effects, including diarrhea, tremors, and hypothermia, 5HTP must be administered slowly and continuously (Jacobsen et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). These adverse effects likely result from bypassing the intrinsic regulatory function of TPH, which provides a finely tuned 5HTP supply according to demand.\u003c/p\u003e\n \u003cp\u003eOur threshold phenomenon (~\u0026thinsp;20 mg/kg in mice) represents an inflection point whereby SP dosage transitions from peripheral BH\u003csub\u003e4\u003c/sub\u003e production (release effect only) to direct SP delivery to brain cells (enabling synthesis enhancement). Understanding this mechanism rationalizes previous BH\u003csub\u003e4\u003c/sub\u003e-related findings and represents a paradigm shift from empirical trial-and-error to rational, mechanism-based BH\u003csub\u003e4\u003c/sub\u003e replacement.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eImplications for specific BH\u003csub\u003e4\u003c/sub\u003e deficiencies:\u0026nbsp;\u003c/strong\u003eSP administration requires intact salvage pathway enzyme activity. In SPR deficiency, SP cannot be converted to BH\u003csub\u003e4\u003c/sub\u003e, rendering it ineffective. Although alternative pathways (carbonyl reductase and DHFR) provide limited BH\u003csub\u003e4\u003c/sub\u003e in the liver, their brain activity remains unclear (Bonafe et al., \u003cspan class=\"CitationRef\"\u003e2001\u003c/span\u003e). DHPR deficiency presents a different challenge, as it impairs BH\u003csub\u003e4\u003c/sub\u003e utilization rather than production. BH\u003csub\u003e4\u003c/sub\u003e utilization requires functional DHPR. Paradoxically, DHPR-knockout mice exhibit significant levels of brain BH\u003csub\u003e4\u003c/sub\u003e (Xu et al., \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e), confirming that BH\u003csub\u003e4\u003c/sub\u003e availability alone is insufficient without functional recycling. These mechanistic constraints define patient populations that would not benefit from SP therapy, underscoring the importance of enzymatic profiling before initiating treatment.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e4.5 Clinical Significance and Therapeutic Potential\u003c/h2\u003e\n \u003cp\u003eThe enhancement of brain BH\u003csub\u003e4\u003c/sub\u003e mediated by SP presents unique advantages for treating monoaminergic disorders. By elevating intraneuronal BH\u003csub\u003e4\u003c/sub\u003e levels above threshold levels, SP simultaneously enhances the synthesis of all monoamines\u0026mdash;serotonin, dopamine, and norepinephrine\u0026mdash;because of their common dependence on BH\u003csub\u003e4\u003c/sub\u003e-dependent hydroxylases. This multi-system approach contrasts with current single-target strategies, such as selective serotonin reuptake inhibitors (SSRIs), and may more adequately address complex disorders, such as major depression, which involves multiple neurotransmitter dysregulations.\u003c/p\u003e\n \u003cp\u003eOur approach addresses a fundamental limitation of current therapies: while SSRIs increase synaptic serotonin (5-HT\u003csub\u003eext\u003c/sub\u003e), they cannot overcome the reduced synthesis capacity in individuals with diminished TPH activity. Although 5-HT\u003csub\u003eext\u003c/sub\u003e is only approximately 1/10,000th of the 5-HT in tissues, its turnover is extremely rapid, making its total traffic comparable to that of released vesicles. Its sustained release is limited by the size of the vesicular pool and, therefore, the capacity to produce 5-HT (Calcagno et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e). Furthermore, this pool is maintained by rapid turnover throughout the brain as a single compartment with \u003cem\u003eT\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e \u0026asymp; 1 h (Carlsson et al., \u003cspan class=\"CitationRef\"\u003e1972\u003c/span\u003e). By enhancing TPH activity while preserving enzymatic regulatory mechanisms, SP increases serotonin production capacity, providing resilience against depletion during high-demand states (e.g., stress).\u003c/p\u003e\n \u003cp\u003eFor other conditions, the biphasic response pattern facilitates personalized medicine approaches: low-dose (monoamine release) versus high-dose (enhanced synthesis) can be tailored to specific pathophysiological conditions. Regional heterogeneity at low doses versus uniform enhancement at high doses suggests the potential for either targeted or global therapeutic strategies, depending on clinical needs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e4.6 Limitations and Future Directions\u003c/h2\u003e\n \u003cp\u003eHowever, some limitations must be considered before our findings can be clinically applied. \u003cem\u003ePharmacokinetic optimization\u003c/em\u003e: Our single-dose studies revealed rapid SP clearance (urinary excretion within 1 h; (Ohashi et al., \u003cspan class=\"CitationRef\"\u003e2024\u003c/span\u003e) [\u003cstrong\u003epreprint\u003c/strong\u003e]). Future studies should explore repeated dosing strategies to maintain therapeutic brain BH\u003csub\u003e4\u003c/sub\u003e levels. \u003cem\u003eRoute-dependent thresholds\u003c/em\u003e: The ~\u0026thinsp;20 mg/kg threshold (mouse, intraperitoneal) may differ with alternative administration routes. Human translation requires comprehensive pharmacokinetic/pharmacodynamic studies that involve multiple delivery methods. \u003cem\u003eCatecholaminergic effects\u003c/em\u003e: Although we focused on serotonergic parameters, the effects of SP on dopamine and norepinephrine synthesis via TH enhancement remain uncharacterized. \u003cem\u003eFunctional outcomes\u003c/em\u003e: Behavioral and cognitive consequences of SP-induced neurochemical changes require systematic evaluation using validated paradigms relevant to monoaminergic function. \u003cem\u003eDrug interactions\u003c/em\u003e: Potential synergistic or antagonistic effects with existing monoaminergic therapies (SSRIs, MAO inhibitors) require investigation before clinical application.\u003c/p\u003e\n \u003cp\u003eAddressing these limitations will refine dosing strategies, identify optimal patient populations, and establish the position of SP within existing therapeutic frameworks for monoaminergic disorders.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e5-HT, serotonin; 5-HIAA, 5-hydroxy-indoleacetic acid; 5HTP, 5-hydroxy-L-tryptophan; 6R-BH\u003csub\u003e4\u003c/sub\u003e, 6R-tetrahydrobiopterin; AADC, aromatic-L-amino acid decarboxylase; BBB, blood\u0026ndash;brain barrier; BH\u003csub\u003e2\u003c/sub\u003e, 7,8-dihydrobiopterin; BH\u003csub\u003e4\u003c/sub\u003e, tetrahydrobiopterin; DHFR, dihydrofolate reductase; DHPR, dihydropteridine reductase; MAO, monoamine oxidase; qBH\u003csub\u003e2\u003c/sub\u003e, quinonoid dihydrobiopterin; SP, sepiapterin; TH, tyrosine hydroxylase; TPH, tryptophan hydroxylase\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest Statement\u003c/h2\u003e\u003cp\u003eH.H. declares no current conflicts of interest. In the past, H.H. served as an advisor to Shiratori Pharmaceutical Co., Ltd (2013), was a member of advisory board for Censa Pharmaceuticals Inc. (2016\u0026ndash;2021), and holds stock options in Censa Pharmaceuticals Inc. (now acquired by PTC Therapeutics). Intellectual property rights in PCT application WO2011/132435, invented by H.H. and S.A. were assigned to Censa Pharmaceuticals Inc. (acquired by PTC Therapeutics). A.O., H.M. and S.A. declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e\u003cp\u003eA.O. and H.H.: Conceptualization, Methodology, Investigation, Formal analysis, Writing - Original Draft, Visualization; H.M.: Formal analysis, Validation; S.A.: Resources, Project administration, Funding acquisition. All authors reviewed and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis research was supported by Shiratori Pharmaceutical Co., Ltd. (2013), and the Dental Research Center, Nihon University School of Dentistry (DRC(B)-2023-1, DRC(B)-2024-1). We are grateful to Prof. Kazuhiro Nakamura of the Applied Health Sciences Unit, Department of Medical Laboratory Science, Gunma University, for his valuable advice regarding the brain dissection protocol. We also thank Prof. Tomihisa Takahashi of the Department of Anatomy, Nihon University School of Dentistry, and Dr. Tomonori Harada of the Department of Anatomy and Functional Morphology, Nihon University School of Medicine, for their kind support of this research.\u003c/p\u003e\u003ch2\u003eData Availability Statement\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBonafe, L., Thony, B., Penzien, J. M., Czarnecki, B., \u0026amp; Blau, N. 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Chem.,\u0026nbsp;\u003c/em\u003e253(19): 6673-6679.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eWerner, E. R., Blau, N., \u0026amp; Th\u0026ouml;ny, B. (2011). \u0026quot;Tetrahydrobiopterin: Biochemistry and pathophysiology.\u0026quot; \u003cem\u003eBiochem. J.,\u0026nbsp;\u003c/em\u003e438(3): 397-414. doi:doi: 10.1042/BJ20110293\u003c/li\u003e\n \u003cli\u003eWinn, S. R., Scherer, T., Th\u0026ouml;ny, B., \u0026amp; Harding, C. O. (2016). \u0026quot;High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).\u0026quot; \u003cem\u003eMol. Genet. Metab.,\u0026nbsp;\u003c/em\u003e117(1): 5-11. doi:10.1016/j.ymgme.2015.11.012\u003c/li\u003e\n \u003cli\u003eXu, F., Sudo, Y., Sanechika, S., Yamashita, J., Shimaguchi, S., Honda, S., Sumi-Ichinose, C., Mori-Kojima, M., Nakata, R., Furuta, T., Sakurai, M., Sugimoto, M., Soga, T., Kondo, K., \u0026amp; Ichinose, H. (2014). \u0026quot;Disturbed biopterin and folate metabolism in the Qdpr-deficient mouse.\u0026quot; \u003cem\u003eFEBS Lett.,\u0026nbsp;\u003c/em\u003e588(21): 3924-3931. doi:10.1016/j.febslet.2014.09.004\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Nihon University","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":"Deficiency, Pharmacokinetics, Replenishment, Sepiapterin, Serotonin, Tetrahydrobiopterin","lastPublishedDoi":"10.21203/rs.3.rs-7300477/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7300477/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSepiapterin (SP) serves as a rate-limiting precursor of tetrahydrobiopterin (BH₄), an essential cofactor for tryptophan hydroxylase (TPH) and tyrosine hydroxylase, which are rate-limiting enzymes in the synthesis of brain monoamines. This study investigated the enhancement of brain serotonin metabolism through peripheral SP administration using comprehensive, \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e methodologies. In serotonin-producing RBL2H3 cells, SP exhibited a 21-fold greater cellular uptake efficiency compared to 6R-BH₄, leading to enhanced intracellular BH₄ levels and TPH activation. Kinetic analysis indicated cooperative TPH behavior (Hill coefficient\u0026thinsp;=\u0026thinsp;2.1) with an apparent \u003cem\u003eK\u003c/em\u003e\u003csub\u003eM\u003c/sub\u003e of 18.1 \u0026micro;M, which closely aligned with the endogenous BH₄ levels. In C57BL/6J mice, systemic SP administration demonstrated a notable biphasic dose\u0026ndash;response pattern with a distinct threshold at approximately 20 mg/kg, delineating two mechanistically distinct regimes. Below this threshold, SP does not reach the brain; however, peripherally-generated BH₄ enters the brain parenchyma but remains predominantly extracellular, stimulating region-specific serotonin release without enhancing synthesis, manifesting as decreased 5-HIAA in brainstem serotonergic nuclei while increasing it in projection areas. Above the threshold, SP directly penetrates the blood\u0026ndash;brain barrier and enters brain cells, elevating intracellular BH₄ levels and enhancing TPH activity, thereby producing uniform 5-HIAA increases across all brain regions. These findings establish SP as an effective strategy for enhancing brain serotonin synthesis through targeted intracellular BH₄ elevation, addressing the fundamental limitations of 6R-BH₄ supplementation that have persisted since the pioneering work of Niederwieser in the 1980s. This mechanistic breakthrough suggests a substantial therapeutic potential for various monoaminergic disorders.\u003c/p\u003e","manuscriptTitle":"Sepiapterin Enhances Brain Tetrahydrobiopterin BH4-Dependent Serotonin Synthesis with Regional Specificity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-11 06:14:28","doi":"10.21203/rs.3.rs-7300477/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":"9f00b32b-9a87-492f-b5b8-ad016fc016fe","owner":[],"postedDate":"August 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52683645,"name":"Cellular \u0026 Molecular Neuroscience"},{"id":52683646,"name":"Drug Discovery, Design, \u0026 Development"}],"tags":[],"updatedAt":"2025-08-11T06:14:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-11 06:14:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7300477","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7300477","identity":"rs-7300477","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}