Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental condition characterized by impaired sociability, repetitive behaviors, and communication deficits. Animal models have been instrumental in elucidating the mechanisms underlying ASD, with prenatal exposure to valproic acid (VPA) being one of the most widely validated approaches. However, most studies rely on intraperitoneal administration, which poorly reflects human exposure. Here, we investigated the effects of oral prenatal VPA exposure in Wistar rats, focusing on behavioral outcomes, biochemical alterations, and sex-dependent differences. Pregnant females received VPA (500 mg/kg) by gavage on gestational days 11-13, and offspring were monitored from neonatal to juvenile stages. VPA-exposed pups exhibited delayed physical maturation, including postponed eye opening, tooth eruption, and locomotor development, along with reduced body weight gain. In the juvenile phase, VPA impaired sociability, reduced exploratory activity, and increased repetitive self-grooming. Importantly, behavioral effects were sex-specific: males showed more pronounced deficits in social interaction, whereas females exhibited stronger stereotyped and anxiety-like behaviors. Biochemical assays revealed elevated malondialdehyde (MDA) and nitrite levels, consistent with oxidative and nitrosative stress, especially in the hippocampus and PFC. Additionally, VPA-exposed females showed a marked reduction in hippocampal glutathione (GSH), while males exhibited increased glutamate and γ-aminobutyric acid (GABA) levels in the PFC, indicating disrupted excitatory/inhibitory balance. Collectively, our findings demonstrate that oral VPA administration induces autism-like phenotypes and region-specific neurochemical alterations in a sex-dependent manner. This study reinforces the translational validity of the oral VPA model and identifies oxidative stress and neurotransmitter imbalance as potential biomarkers for ASD pathophysiology and therapeutic intervention.
Neurochemical imbalances, including elevations of the tryptophan metabolite kynurenic acid (KYNA), an endogenous antagonist of glutamatergic and cholinergic receptors, are linked to cognitive and sleep disturbances in psychiatric and neurocognitive disorders. Therapeutic strategies to reduce brain KYNA by inhibiting kynurenine aminotransferase II (KAT II) are under investigation. However, few studies consider time as a biological variable, despite recent evidence that the time of day can affect brain metabolism and drug effectiveness. Therefore, we explore the hypothesis that KYNA formation and synthesis inhibition change throughout the day. Using rats of both sexes, we measured basal KYNA levels and the effects of kynurenine (100 mg/kg, i.p.) to stimulate de novo KYNA, and/or PF-04859989 (KAT II inhibitor, 30 mg/kg, s.c.) at the beginning of light or dark phases. Microdialysis was used to assess extracellular KYNA in the dorsal hippocampus, and ex vivo assays evaluated KAT I and KAT II enzyme activity in separate animals. Additionally, we examined KYNA levels and the effect of PF-04859989 during acute sleep deprivation in male rats. Regardless of phase, PF-04859989 reduced basal KYNA levels in male but not female rats, yet it reduced kynurenine-stimulated KYNA synthesis in both sexes, demonstrating a context-specific action in female rats. Importantly, we observed a novel effect of phase in males, as kynurenine-induced KYNA synthesis and its inhibition by PF-04859989 were greater during the dark phase than during the light phase. Ex vivo, male KAT II activity was higher, and PF-04859989 was more effective, in the dark than in the light phase, suggesting that properties of the KAT II enzyme itself fluctuate with time of day. Finally, sleep deprivation increased extracellular KYNA levels in the light phase, and PF-04859989 fully ameliorated this increase. Overall, our findings highlight the need to consider time-dependent factors when developing therapies impacting KYNA synthesis.
Amacrine cells (ACs) comprise a heterogeneous class of inhibitory neurons in the vertebrate retina, exhibiting morphological and functional complexity rivaling that of cortical interneurons. Here, we integrate single-cell and single-nucleus transcriptomic atlases from 24 vertebrate species to reconstruct the evolutionary origins of this extreme diversity. We identify 42 orthologous AC types (oACs), most of which exhibit a one-to-one correspondence across amniotes and, in many cases, across vertebrates. While core molecular identities are conserved, AC types vary in abundance and gene expression across species, likely reflecting adaptations to distinct visual ecologies. AC diversity scales with that of retinal ganglion cells (RGCs), indicative of coevolution. Finally, we suggest that ACs arose from an AC-RGC hybrid precursor, with glycinergic ACs diverging early in vertebrate evolution, followed by a bifurcation between RGCs and GABAergic ACs. Together, these findings establish a unified evolutionary framework for understanding the diversity, development, and function of a class of inhibitory neurons across vertebrates.
Indole-tetrahydroazepine derivatives of iboga alkaloids (ibogalogs) exhibit antidepressant, anxiolytic, promnesic, and antineuropathic effects, mainly via serotonergic targets. However, their physicochemical properties relevant to redox biology, including antioxidant activity and membrane stability, have remained poorly characterized. We investigated the antioxidant properties of three ibogalogs: ibogaminalog (DM506), ibogainalog (IBG), and tabernanthalog (TBG), using model lipid membranes (liposomes), human erythrocytes, and rat hippocampal and cortical synaptosomes exposed to AAPH-induced oxidative stress. Physicochemical descriptors, bond dissociation enthalpies, and ionization potentials were also calculated to assess membrane interactions and the antioxidant potential. All ibogalogs protected erythrocytes by reducing hemolysis, potassium efflux, and malondialdehyde levels, with the strongest effects observed for TBG; none induced hemolysis or K+ efflux at 0.01-10 μM. Ibogalogs also decreased lipid peroxidation in rat hippocampal and cortical synaptosomes. In liposomal systems, TBG showed the highest efficacy against lipid-peroxyl-radical-induced peroxidation, whereas DM506 and IBG mainly slowed autoxidation. Theoretical analysis indicated that the methoxy group substitution critically influences bond dissociation enthalpies, radical delocalization, and antioxidant potency. This first physicochemical characterization of antioxidant properties of ibogalogs enhances the understanding of their membrane-protective actions, complementing their neuromodulatory profiles.
HDACs play important roles in various neurological disorders. Unlike other HDAC subtypes that have been extensively studied in neurodegenerative diseases, HDAC8 has only recently emerged as a regulator in the central nervous system. In this study, we designed and successfully radiosynthesized [11C]HYF031 as a potential HDAC8 radioligand with acceptable radiochemical yield and purity. In vitro autoradiography and self-blocking dynamic PET/CT studies in C57BL/6 mice demonstrated specific binding of [11C]HYF031 to HDAC proteins in the brain. Dynamic PET imaging further showed measurable brain uptake and pharmacokinetic properties compatible with PET imaging. Biodistribution studies indicated that the tracer is primarily metabolized through the hepatobiliary system. Overall, these results suggest that [11C]HYF031 is a promising lead tracer for HDAC8 imaging.
Pathological aggregation of α-synuclein is a hallmark of synucleinopathies such as Parkinson's disease, where fibrillar α-synuclein aggregates drive neurodegeneration. Here, we aimed to identify small molecules capable of disassembling fibrillar α-synuclein aggregates by screening a natural product library using a plasmonic nanoparticle amyloid corona platform. Candidates were further ranked based on key physicochemical properties (molecular weight, solubility, and lipophilicity) associated with cell permeability and potential central nervous system accessibility. Through this analysis, angelic acid emerged as the top candidate. Physicochemical characterization, including circular dichroism, Fourier-transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy, demonstrated that angelic acid disrupts β-sheet-rich conformations and fragments α-synuclein fibrils. Molecular docking analysis suggested potential interactions of angelic acid with β-sheet interface regions across multiple α-synuclein fibril polymorphs. In a bimolecular fluorescence complementation cell model, angelic acid reduced intracellular α-synuclein accumulation by up to 91.4% at 100 μM. In addition, angelic acid alleviated α-synuclein fibril-induced cytotoxicity by 34.1%, demonstrating both reduced cellular α-synuclein levels and attenuation of α-synuclein fibril-induced cytotoxicity. Collectively, these findings suggest that angelic acid is a pathological α-synuclein-targeting lead compound for synucleinopathies, highlighting the need for further in vivo evaluation in synucleinopathy models.
A well-regulated metabolite concentration in biological fluids is essential for maintaining a normal physiological homeostasis. However, under conditions of metabolic imbalance, certain metabolites can accumulate and undergo self-assembly into amyloid-like supramolecular structures that may disrupt cellular functions. Understanding the aggregation behavior of such metabolites is therefore important for elucidating the molecular mechanisms underlying metabolite-associated disorders. In this study, we investigate the self-assembly properties of phenylalanine-derived metabolites, namely, phenylacetic acid (PA), phenyllactic acid (PL), and phenylpyruvic acid (PP). Experimental characterization revealed that these metabolites form diverse supramolecular assemblies near physiological pH. The amyloid-like nature of these structures was confirmed using established amyloid-binding dyes, including thioflavin T (ThT), Nile red, and Congo red. Density functional theory (DFT) calculations were employed to analyze the intermolecular interactions responsible for molecular association. In addition, 100 ns molecular dynamics simulations of multimonomer systems in explicit water demonstrated spontaneous aggregation of the metabolites, revealing distinct aggregation propensities. PP exhibited the strongest aggregation behavior, forming large supramolecular clusters (average cluster size = 18.4 ± 0.7 monomers at 100 ns), followed by PA (15.2 ± 0.9), whereas PL showed comparatively weaker association (9.7 ± 1.1). Cytotoxicity assays and flow cytometry (FACS) analysis further revealed that the self-assembled structures of PA and PP induce apoptotic cell death similar to that of phenylalanine aggregates, whereas PL displayed comparatively higher cellular compatibility even at elevated concentrations. These findings highlight distinct aggregation behaviors among phenylalanine-derived metabolites and provide new insights into their potential role in the molecular pathology of phenylketonuria (PKU), thereby contributing to a deeper understanding of metabolite-driven aggregation phenomena in neurochemical disorders.
The multifactorial origin of Alzheimer's disease (AD) is currently being addressed with the development of combination therapy or multitarget directed ligands. If the conventional approach of targeting acetylcholinesterase (AChE) for AD treatment has limitations, it could offer opportunities for a polypharmacological approach by designing covalent pseudoirreversible prodrugs inspired by rivastigmine's mechanism of action. This study focuses on introducing aminated drugs to the rivastigmine carbamate moiety, namely, fluoxetine and memantine, which have shown synergy with cholinesterase inhibition. These innovative carbamates target sustained drug release through covalent pseudoirreversible cholinesterase inhibition, strategically balancing inhibitory potency, selectivity, mechanism, and reactivation kinetics. This comprehensive approach demonstrates the potential of targeting ChE via a covalent mechanism and provides valuable insights into the structure-activity relationships of these derivatives. Interestingly, this study provides a useful biochemical toolbox for characterizing pseudoirreversible cholinesterase carbamate-type inhibitors. The most promising compound was evaluated in in cellulo and in vivo AD models, highlighting the potential of polypharmacological interventions as innovative and multifaceted anti-AD drugs.
Purpose: The essential oil of Hyptis crenata (EOHc) contains several terpenes, many of which have anxiolytic-like activity. Thus, the aim of this study was to extract, analyze the chemical composition, and evaluate the anxiolytic-like effect of EOHc in mice using experimental approaches (behavioral parameters) and in silico studies. Methods: Mice were divided into seven experimental groups: Group I (no stress); Group II, stress only (no treatment); Group III, Tween 80 (stress and 0.1%, vehicle); Group IV, Tween 80 (no stress and 0.1%, vehicle); Group V, mirtazapine (stress and 30 mg/kg); Group VI, citalopram (stress and 10 mg/kg); Group VII, essential oil of H. crenata (stress and EOHc, 100 mg/kg); Group VIII, essential oil of H. crenata (no stress and EOHc, 100 mg/kg); Group IX, essential oil of H. crenata (stress and EOHc, 300 mg/kg); and Group X, essential oil of H. crenata (no stress and EOHc, 300 mg/kg);. After eight stress days, the behavioral tests (open field, elevated plus maze, and RotaRod) were started. Furthermore, docking and molecular dynamics analysis were used. To evaluate the safe administration of EOHc, water consumption, food consumption, and weight parameters were evaluated during 7 days of treatment. Results: The oil yield was 1.0-1.5%. Chromatography revealed that the top 5 constituents were caryophyllene V1, azulene, α-pinene, bornanone, and viridiflorene. All the treatments reduced the crossover and the rearing (p-value ≤ 0.05; ANOVA; Tukey's post hoc test). During grooming time, neither stress nor treatments induced any changes. The stress altered the number of entries and the time spent in both open arm and closed effect that was reversed by all treatments except mirtazapine (p ≤ 0.05; ANOVA; Tukey's test). There was no significance in the evaluation of water consumption, food consumption, and weight. With the exception of the Tween group, the RotaRod Test showed no significance between the control, 100 mg/kg EOHc, and 300 mg/kg EOHc groups. Conclusion: It can be concluded that EOHc presents an anxiolytic effect experimentally, with the probable inhibition of Apo SERT, due to the terpenoid constituents present in the oil.
Parkinson's disease (PD) is a prevalent neurodegenerative movement disorder characterized by bradykinesia, rigidity, and resting tremor, progressing insidiously over time. Central to its pathophysiology is the degeneration of dopaminergic neurons in the substantia nigra, leading to a significant decrease in striatal dopamine (DA) levels. This dopaminergic deficit disrupts basal ganglia circuitry, impairing motor function and contributing to the core symptoms of the disease. While the etiology of PD remains incompletely understood, a combination of genetic predispositions and environmental exposures has been implicated. Beyond dopaminergic dysfunction, emerging evidence suggests that other neurotransmitter systems, including noradrenergic, serotonergic, cholinergic, glutamatergic, and γ-aminobutyric acidergic (GABAergic) pathways, are also involved in disease progression and symptom heterogeneity. Pathological hallmarks such as α-synuclein (α-syn) misfolding and Lewy body (LB) formation, along with mitochondrial dysfunction, oxidative stress, and neuroinflammation, further exacerbate neurodegeneration and neurotransmitter imbalances. Despite advances in symptomatic treatment, current therapies primarily target DA deficiency and fail to reverse neurodegenerative processes. The involvement of multiple neurotransmitter systems highlights the complex neurochemical landscape of PD and underscores the need for multifaceted therapeutic strategies. Understanding the broader role of neurotransmitters in PD pathogenesis offers promising avenues for disease-modifying interventions and improved symptom management. This review summarizes the recent findings on the contribution of various neurotransmitters to PD, emphasizing their potential as targets for future therapeutic development. By integrating the current literature, we aim to provide a comprehensive overview of neurotransmitter involvement in PD and its implications for advancing treatment paradigms.
γ-Secretase (GS) is a key therapeutic target in Alzheimer's disease because it catalyzes amyloid-β production. To reduce toxic amyloid-β species, several γ-secretase modulators (GSMs) have been developed. Among these, GSM-1, a piperidine carboxylic acid-based modulator, has shown potent activity in preclinical studies; however, its binding site and molecular mechanism remain unclear. Here, a molecular dynamics protocol inspired by the Wrap-'N'-Shake approach was used to investigate how the axial and equatorial conformations of the piperidine nitrogen in GSM-1 interact with GS's presenilin subunit. Both conformations formed stable interactions with presenilin TM1 and TM5. The carboxylate group established persistent interactions with R220 in TM5, while the trifluoromethylphenyl group engaged residues K76, K80, V87, and L91 in TM1. These findings provide the first model of GSM-1 engagement with GS, identify the key GSM-1 chemical groups involved in recognition, and establish a mechanistic foundation for the rational design of next-generation GSM analogs.
Evaluation of data quality always remains labor-intensive, but it is a critical prerequisite in clinical neuroscience research to ensure the reliability and reproducibility of research outcomes. The unavailability of quality control (QC) pipelines on a single platform for multimodal neuroimaging data has been a limiting factor in extensive brain research. Moreover, existing QC pipelines primarily involve MRI-based data only. In this context, we developed a neuroimaging and neurospectroscopy (NINS) QC platform "NINS_QC" for multimodal neuroimaging (MRI, fMRI, DTI, MRS) and neuropsychological data. NINS_QC is a transparent and user-friendly framework that can be distributed as a standalone or in the form of a downloadable executable file. This allows local processing of multimodal neuroimaging data and is well-suited for environments with stringent data privacy requirements. The presence of metrics-based analysis, along with visual-based assessment, has added one more attribute to this platform.
Traumatic brain injury (TBI) triggers complex secondary pathological mechanisms, including neuroinflammation, oxidative stress, and apoptosis, contributing to long-term cognitive and motor deficits. This study investigates the neuroprotective potential of Clemizole, a known TRPC5 inhibitor, in a weight-drop rat model of TBI. Target prediction analyses using Swiss Target Prediction and CTD databases identified 159 overlapping genes between Clemizole and TBI. Protein-protein interaction network and hub gene analyses highlighted key proteins, such as TNF-α, CASP3, MMP-9, and TRPC5, implicating them in TBI pathogenesis. KEGG pathway enrichment revealed Clemizole-targeted pathways, including PI3K-Akt, TNF signaling, and apoptosis. After TBI, behavioral assessments showed that Clemizole significantly improved neurological scores, grip strength, locomotor activity, and spatial learning deficits. Biochemical assays revealed that Clemizole dose-dependently reduced nitrite and MDA levels while restoring GSH, indicating attenuation of oxidative stress. H&E (hematoxylin and eosin) and cresyl violet staining confirmed reduced neuronal degeneration and preserved cortical integrity. Clemizole also downregulated inflammatory cytokines and glial markers (Iba-1 and GFAP), alongside restoring BBB integrity via upregulation of tight junction proteins and suppressing MMP-9 expression. Furthermore, Clemizole activated the PI3K-Akt signaling pathway, decreasing the expression of pro-apoptotic proteins (Bax, caspase-9 and caspase-3) and restoring Bcl-2 levels. Importantly, Clemizole decreased TRPC5 expression and attenuated CHOP-mediated ER stress, suggesting a mechanistic link between TRPC5 inhibition and PI3K-Akt-mediated neuroprotection. Collectively, these findings demonstrate that Clemizole confers multifaceted neuroprotection following TBI by targeting TRPC5-mediated calcium dysregulation, restoring PI3K-Akt signaling, and attenuating oxidative, inflammatory, and apoptotic cascades. This study identifies Clemizole as a promising therapeutic candidate for mitigating secondary brain injury and promoting functional recovery after TBI.
Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are linked to familial amyotrophic lateral sclerosis (ALS), yet the identity of the toxic molecular species remains unclear. We investigated the relationship between protein misfolding and pathogenicity by expressing GFP-tagged wild-type and mutant SOD1 (A4V, H46R, G93A) in mouse hippocampal HT22 cells. Western blotting under nonreducing conditions suggested that A4V, associated with rapid disease progression, was largely depleted of properly folded soluble SOD1 and instead produced highly destabilized soluble species. In contrast, H46R, associated with a milder phenotype, showed a moderate reduction in properly folded soluble SOD1 and generated partially folded/native-like conformers. G93A exhibited biochemical characteristics intermediate between those of A4V and H46R. A4V also showed a pronounced loss of GFP fluorescence, indicating severe structural destabilization; the extent of fluorescence loss in A4V, G93A, and H46R broadly correlated with clinical severity. Neither CuATSM nor ebselen─targeting metal binding and disulfide formation, respectively─rescued fluorescence, suggesting broader defects in SOD1 maturation. Nevertheless, both compounds inhibited ferroptosis, a nonapoptotic form of cell death characterized by iron-dependent lipid peroxidation, in HT22 cells, indicating alternative neuroprotective mechanisms. These findings identify destabilized soluble SOD1 species as a key toxic entity in ALS and highlight the utility of GFP-tagged constructs for evaluating folding status and screening therapeutic candidates.
While antisense oligonucleotide (ASOs) therapies have emerged as promising tools to modulate gene expression in neurological diseases, these new agents face challenges in the context of the central nervous system, particularly regarding distribution and duration of action. Translational approaches that enable the tracking of ASO effects in the whole brain are therefore needed to guide their preclinical and clinical development. In the present study, we used PET imaging as a translational tool to study the pharmacodynamics of an ASO targeting a metabotropic receptor, namely, the dopamine D2 receptor. We selected an ASO sequence directed toward D2 mRNA in the rat brain and optimized its chemical backbone by a gapmer design. This anti-D2R ASO was administered directly into the striatum of adult rats via intracerebral stereotaxic injection at different doses. The longitudinal pharmacodynamic effects of the ASO were assessed using successive PET acquisitions with the D2R radiotracer [11C]raclopride to quantify changes in D2R receptor expression in the striatum. Our PET findings indicated a reduction of D2R availability from 3 weeks to 10 weeks postinjection. The apomorphine-induced rotation test confirmed that the postsynaptic striatal dopaminergic imbalance was behaviorally relevant and persisted for three months after the ASO injection. This work provides novel insights into the potential of PET neuroimaging to explore the in vivo efficacy and longevity of modified ASOs delivered directly into the brain, opening translational applications.
Norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) are key neurotransmitters that regulate mood, cognition, and neurochemical balance. Abnormal levels of these molecules are linked with several neuropsychiatric disorders, underscoring the need for highly sensitive and selective detection methods. In this study, a hierarchical plasmonic nanocomposite, denoted as zeolitic imidazolate framework-67 (ZIF-67)@gold nanoparticles (Au)@polydopamine (PDA)@silver nanoparticles (AgNPs) supported on PDA/polyethyleneimine (PEI)-coated glass (glass@PDA/PEI), is reported, which features a metal-organic framework (MOF) template and dual-metal plasmonic coupling between Au (20.6± 5.1 nm) and Ag (5.5 ± 1.8 nm) mediated by a PDA interlayer. This platform demonstrates an excellent surface-enhanced Raman scattering (SERS) performance, achieving detection limits (LOD) of 1.6 × 10-11 M for NE and 1.3 × 10-12 M for 5-HT, along with reliable linearity across physiologically relevant concentration ranges. Notably, the SERS system was successfully employed for the first time to detect 5-HT levels in human plasma samples from 25 healthy individuals, yielding an average concentration of 1.9 × 10-8 M and confirming its feasibility for application in real biological matrices. This work pioneers a SERS-based quantitative platform for detecting 5-HT in human plasma, demonstrating its promise for the early diagnosis and monitoring of neuropsychiatric diseases associated with dysregulated 5-HT levels.
Axonal degeneration (AxD) is a defining feature of many neurodegenerative disorders, with SARM1 functioning as a key pro-degenerative NADase that becomes activated through conformational change. Here, we identify a critical regulatory mechanism mediated by the SARM1-specific (SS) loop within its TIR domain. Using mutagenesis, activity assays, and molecular dynamics simulations, we demonstrate that dynamic conformational transitions of the SS loop, particularly the flipping of His640, are essential for NAD recruitment and catalytic site assembly. Zinc ions inhibit SARM1 activity by coordinating with a conserved C2H2 motif in the SS loop, thereby restricting His640 flipping and preventing NAD engagement. Notably, chemical tethering of adjacent cysteine residues within the SS loop similarly suppresses its conformational dynamics, resulting in potent inhibition of catalysis. The essential role of SS loop dynamics uncovered here offers new mechanistic insight and establishes a foundation for developing therapeutic strategies targeting SARM1.
Alzheimer's disease (AD) is a debilitating neurodegenerative disorder characterized by cognitive decline and memory loss. Current treatments offer limited efficacy, necessitating the development of innovative multitarget therapeutic strategies. Here, we present N3,N5-bis(2-(5-methoxy-1H-indol-3-yl)ethyl)-2,6-dimethyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxamide (HCM-01), a novel compound developed to target multiple neurodegenerative pathways implicated in AD. In vitro assays included MTT-based cell viability analyses performed in two complementary experimental settings: primary neuronal cultures and astrocyte-based in vitro cell culture models exposed to glutamate. In primary hippocampal neuronal cultures, glutamate exposure induced a statistically significant reduction in cell viability compared with vehicle-treated controls, consistent with glutamate-induced excitotoxicity. Under these conditions, HCM-01 treatment resulted in a statistically significant improvement in neuronal viability, showing a greater protective effect compared with donepezil and memantine. In contrast, in astrocyte-based in vitro cultures, the applied glutamate concentration did not induce overt cytotoxicity, in line with the intrinsic neuroprotective and glutamate-buffering role of astrocytes. Accordingly, astrocytic experiments were designed to assess functional modulation of glutamate-handling mechanisms rather than cell survival. Western blot analysis in C8-D1A astrocytic cells demonstrated increased expression of excitatory amino acid transporter 2 (EAAT2) following HCM-01 treatment compared with control and reference drug-treated groups, suggesting modulation of astrocyte-mediated glutamate homeostasis. In parallel, redox analyses revealed that HCM-01 improved oxidative/antioxidative balance, as evidenced by increased total antioxidant capacity (TAC) and reduced total oxidant status (TOS), supporting an indirect antioxidant contribution to its functional effects. In vivo behavioral assessment of HCM-01 in a streptozotocin (STZ)-induced Alzheimer's model in female Sprague-Dawley rats demonstrated that administration of HCM-01 at doses of 50 mg/kg orally (oral, P.O. and intraperitoneal, I.P.) and 100 mg/kg (P.O.), significantly improved cognitive and memory functions in the passive avoidance (PA), Morris water maze (MWM), and locomotor activity tests. Moreover, histopathological and immunohistochemical analyses of different hippocampal regions revealed reduced neuronal damage, attenuation of tau pathology, antiamyloidogenic effect, and restoration of cholinergic function. Complementary in silico studies, including molecular docking, molecular dynamics simulations (MDS), and free energy calculations, suggested potential interactions of HCM-01 with the allosteric site of EAAT2. Taken together, these findings suggest that HCM-01 exerts neuroprotective effects against glutamate-induced excitotoxicity in primary hippocampal neurons while additionally modulating glutamatergic homeostasis and redox balance through functional mechanisms in astrocyte-based models, supporting its relevance as a multitarget preclinical candidate for early stage AD mechanisms.
N-Benzyl-derived phenethylamines are highly active (psychedelic) 5-HT2AR (serotonin 2A receptor) agonists used as biochemical tools and on the drug market. The impact of adding an N-benzyl substituent to the tryptamine core structure has scarcely been studied. A recent systematic exploration of N-benzyl-substituted (5-MeO-)tryptamines revealed a surprisingly low activity in a Ca2+ mobilization assay for certain analogues. Considering the increased number of reports on biased agonism at the 5-HT2AR, with particular examples in the group of phenethylamines containing an N-benzyl substituent, a series of 16 of these (5-MeO-)tryptamine derivatives (14 of which containing an N-benzyl group) was evaluated in two additional assays. Specifically, two highly similar yet complementary NanoBiT assays, monitoring the recruitment of β-arrestin 2 (βarr2) or miniGαq to the 5-HT2AR, were applied. The functional data allowed assessment of the impact of different substituents on the compounds' 5-HT2AR activity: (i) the introduction of an N-benzyl group on the (5-MeO-)tryptamine core; (ii) the substitution of the N-benzyl at different positions; and (iii) the presence of a 5-MeO group on the tryptamine core structure. Interestingly, the N-benzyl-substituted (5-MeO-)tryptamines were more active in βarr2 than in the miniGαq recruitment assay, and biased agonism was assessed by both quantitative and qualitative methods. Aside from the estimation of structure-activity relationships and biased agonism, the current data set allowed for a comparison of the functional data between different assays, hence providing a better understanding of the translatability of results from one assay to the other.