Inosine, a purine nucleoside, has shown potential therapeutic effects in the treatment of Alzheimer's disease (AD) and depression. It acts as a neuroprotective agent by promoting axonal regeneration and enhancing neuronal survival. Studies have indicated that Inosine can improve cognitive functions and alleviate depressive symptoms by modulating neurotransmitter levels and reducing oxidative stress. Additionally, inosine has been found to enhance synaptic plasticity and neurogenesis, which are critical for cognitive and emotional health[1,2].
In our research, we have demonstrated through molecular docking simulations and molecular interaction experiments that inosine can bind to the neuronal nitric oxide synthase (nNOS) receptor. Current antidepressant drugs on the market suffer from insufficient targeting, significant side effects, and delayed onset of action. Research has found that in the dorsal raphe nucleus (DRN), serotonin transporter (SERT) is highly colocalized with nNOS, but this colocalization is largely absent in the postsynaptic region[2]. After chronic stress, SERT-nNOS coupling in the DRN increases, leading to reduced membrane localization of SERT, increased intercellular serotonin concentration, activation of serotonin autoreceptors, and enhanced negative feedback inhibition of serotonergic neuron firing, resulting in reduced serotonin concentration in the postsynaptic synaptic cleft and inducing depression. The rapid antidepressant mechanism is facilitated by a strategic intervention that decouples the serotonin transporter (SERT) from neuronal nitric oxide synthase (nNOS), while simultaneously augmenting the presence of SERT on the membrane of dorsal raphe nuclei (DRN). This dual action results in a diminished level of intercellular serotonin, effectively attenuating the negative feedback loop. Consequently, the firing rate of serotonergic neurons is enhanced, precipitating a swift surge in serotonin levels within the postsynaptic synaptic cleft. This intervention elicits a prompt antidepressant response that is distinct from the desensitization of serotonin autoreceptors, offering a novel and efficacious approach to treating depression.
Therefore, we aim to target the nNOS receptor by using gut microbiota-produced small molecules, which can reach the brain via the gut-brain axis, bind to depression-related nNOS receptors, uncouple SERT from nNOS, and alleviate depression symptoms[3].
[1] Chen, P., Goldberg, D. E., Kolb, B., Lanser, M., & Benowitz, L. I. (2002). Inosine induces axonal rewiring and improves behavioral outcome after stroke. Proceedings of the National Academy of Sciences, 99(13), 9031-9036.
[2] Walker, T. L., & Kempermann, G. (2014). One mouse, two cultures: isolating adult hippocampal neurogenesis in vitro. Frontiers in Neuroscience, 8, 384.
[3] Sun, N., Qin, Y. J., Xu, C., et al. (2022). Design of fast-onset antidepressant by dissociating SERT from nNOS in the DRN. Science, 378(6618), 390-398.
In the context of Alzheimer's disease (AD) precipitated by amyloid-beta (Aβ) protein deposition associated with the APOE4 allele, the identification of targeted intervention strategies remains an ongoing challenge. The inhibition of the LilrB3-APOE4 interaction represents both a significant obstacle and a potential breakthrough in the current therapeutic landscape for AD. Our proposed strategy involves modulating the gut microbiota through the use of microbiome-derived small molecules that specifically bind to the LilrB3 receptor. This approach aims to occupy the LilrB3 receptor sites, thereby competitively inhibiting the binding of APOE4 to these receptors. In the selection of which small molecules can effectively bind to LilrB3 receptors, we propose the use of molecular docking simulation techniques, bioinformatics tools such as AutoDock, and screening of gut microbiota metabolites to identify multiple small molecules with favorable interactions with LilrB3. We employed surface plasmon resonance (SPR) Biacore technology to evaluate the binding affinities between these small molecules and LilrB3 protein, considering the inherent properties and toxicity of the small molecules to select appropriate candidates.
Through the application of molecular docking simulations using software such as AutoDock, in conjunction with surface plasmon resonance (SPR) Biacore assays for molecular interaction analysis, it was conclusively demonstrated that tetrahydrofolate exhibits a strong binding affinity to the LilrB3 receptor. Given the safety profile and non-toxicity of this small molecule, along with its natural production by Lactobacillus plantarum, we propose that tetrahydrofolate represents a promising candidate for mitigating Alzheimer's disease (AD) symptoms through its biosynthesis by Lactobacillus plantarum.Tetrahydrofolate plays a crucial role as a coenzyme in one-carbon metabolism, essential for the proper functioning of the nervous system. It aids in the synthesis of neurotransmitters and DNA methylation, thereby improving cognitive function and mood regulation[1]. Tetrahydrofolate can lower homocysteine levels, reducing oxidative stress and neuroinflammation, offering therapeutic benefits for individuals with Alzheimer's Disease. Additionally, tetrahydrofolate 's role in mood regulation can help address depressive symptoms in AD patients[1,2].
Subsequently, we initiated the design of plasmids. In terms of engineered bacterial selection, we intend to utilize Lactobacillus plantarum L168. Lactobacillus plantarum L168 has been demonstrated to confer significant benefits in regulating gut microbiota andimprove defects of social behvior . Moreover, Lactobacillus plantarum is a harmless probiotic bacterium that does not colonize the human body for an extended period, which greatly aids in controlling the dosage of small molecule drugs within the body. Additionally, probiotic ingestion is more acceptable than drug intake, leading to better compliance among elderly patients.
We initially identified the key enzyme gene sequences responsible for the biosynthesis of tetrahydrofolate in Lactobacillus plantarum by conducting extensive searches on NCBI, UniProt, and various scholarly databases. Through this investigation, we pinpointed the critical enzymes: folK and folE, which are sometimes collectively referred to as folKE in the literature[3]. Subsequently, we successfully integrated these genes into our meticulously designed plasmid, which was equipped with a fail-safe suicide switch.
We then transformed the engineered plasmid into Lactobacillus plantarum L168 to express the tetrahydrofolate. Our aim was to engineer a probiotic strain of Lactobacillus plantarum capable of delivering a small molecule to the brain. Upon ingestion, this probiotic would colonize the intestines and release tetrahydrofolate. Designed to traverse the bloodstream and the gut-brain axis, the molecule tetrahydrofolate would then bind to LilrB3 receptors in the brain. By outcompeting APOE4 for receptor binding, it would help curb amyloid protein deposition, potentially leading to improved Alzheimer's disease symptoms.
[1] Zhang, S.-M., et al. (2002). Folate, vitamin B6, and vitamin B12 intakes in relation to risk of stroke among men. Stroke, 33(1), 196-202.
[2] Reynolds, E. H. (2006). Vitamin B12, folic acid, and the nervous system. The Lancet Neurology, 5(11), 949-960.
[3] Zhu D, Fu Y, Liu F, Xu H, Saris PE, Qiao M. Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000. Microb Cell Fact. 2017;16(1):1. Published 2017 Jan 3. doi:10.1186/s12934-016-0616-2.
