Design:

Tetrahydrofolate (THF) plays a crucial role as a coenzyme in one-carbon metabolism, which is essential for the proper functioning of the nervous system. Studies have indicated that tetrahydrofolate has potential benefits in treating Alzheimer's disease (AD) and depression. It aids in the synthesis of neurotransmitters and DNA methylation, thereby improving cognitive function and mood regulation. Furthermore, tetrahydrofolate can lower homocysteine levels, which helps reduce oxidative stress and neuroinflammation, providing therapeutic benefits for individuals with AD and depression[1,2].

In our research, we have demonstrated through molecular docking simulations and molecular interaction experiments that tetrahydrofolate can effectively target the latest LilrB3 receptor, which is closely associated with APOE4 in AD and represents a pivotal target. By competitively inhibiting APOE4-LilrB3 receptor binding, this strategy addresses the root cause of amyloid protein generation and deposition, significantly advancing AD treatment and prevention and opening up new avenues for engineered bacterial therapy in AD.

We choose Lactobacillus plantarum L168 as the chassis and pSIP403 as the vector. By searching its synthetic pathway, we found the key enzyme folKE and its coding genes folK and folE. Then, we inserted the gene folKE into our designed plasmids to complete the construction and synthesis of plasmids. In view of biosafety, we designed a kill switch which is activated under hypoxic conditions, include a Hypoxia-inducible promoter, a RBS, a coding of SrpR repressor that inhibits transcription of the SrpR-dependent promoter and a terminator.

Build:

The entire plasmid include the coding of tetrahydrofolate-folKE as our target product to bind to target Lilrb3 for the treatment of Alzheimer's disease. And a suicide switch, include a Hypoxia-inducible promoter, a RBS, a coding of SrpR repressor that inhibits transcription of the SrpR-dependent promoter and a terminator. (See more in design safety-suicide switch)

Regrettably, we encountered difficulties when attempting to concatenate the suicide switch with the target gene folKE due to time constraints, which prevented us from successfully constructing the plasmid and transforming it into Lactobacillus plantarum. Consequently, in the subsequent experimental validation, we utilized the plasmid pSIP403-P9-folKE, which expresses only the target gene.

TEST:

According to our experimental procedures, we successfully transformed plasmids into L168 , confirmed through PCR and agarose gel electrophoresis. After culturing L168 , we extracted RNA and measured metabolites from the supernatant. We then conducted reverse transcription and quantitative PCR (qPCR) to quantify gene expression levels in L168 , both with and without the pSIP403-P9-folKE plasmid. The experiment assessed the relative expression levels of two genes using qPCR. The results, shown in the figure 1, indicate that the expression of folK and folE was significantly higher in L168 containing the pSIP403-P9-folKE plasmid compared to the control group (Fig. 1).

Figure 1. Relative Expression Analysis of genes folK and folE by qPCR.

Moreover, we employed Liquid Chromatography-Mass Spectrometry (LC-MS) to analyze THF production in the supernatant of L168 .The results clearly show that THF levels significantly increased in L168 with the pSIP403-P9-folKE plasmid compared to the control group, confirming that the folKE gene was effectively expressed in L168 and produced THF as anticipated (Fig. 2).

Figure 2. Relative levels of THF between two groups by LC-MS.

You can find more information on the Results page.

Learn:

Application of Molecular Docking and Interaction Experiments: The demonstration of tetrahydrofolate's (THF) effectiveness in targeting the LilrB3 receptor, associated with AD pathology, through molecular docking simulations and molecular interaction experiments, provides a scientific foundation for developing novel therapeutic strategies. This approach not only deepens our understanding of the mechanisms underlying AD but also paves the way for innovative treatment modalities.

Utilization of Genetic Engineering: The construction of a plasmid containing the THF synthesis genes (folKE), coupled with a suicide switch mechanism, allows for the design of an engineered bacterial strain that produces THF under specific conditions, such as intestinal hypoxia. This strategy is not only beneficial for AD treatment but also offers new perspectives for the treatment of other diseases.

Experimental Validation: The successful transformation of plasmids into L168 , confirmed through PCR and agarose gel electrophoresis, followed by RNA extraction, metabolite measurement, and quantitative PCR (qPCR) to quantify gene expression levels, validates the increase in gene expression and THF production. These experimental results not only confirm the effectiveness of the genetic engineering strategy but also lay the groundwork for further clinical applications.

Safety and Specificity: The inclusion of a suicide switch ensures the self-destruction of the engineered bacterial strain in non-target environments (e.g., high-oxygen environments outside the intestine), which helps to mitigate potential risks associated with the release of genetically engineered organisms into the environment.

Future Research Directions: This study offers a fresh perspective on AD treatment but also indicates areas that require further investigation, such as the long-term effects of THF in vivo, safety assessments, and potential synergistic effects with other treatment methods.

In future experiments, we will strive to validate the functionality of the hypoxia-inducible suicide switch and continue to explore the protocols for constructing engineered Lactobacillus plantarum L168 strains, endeavoring to successfully integrate the target gene with the suicide switch.

Design:

Inosine, a purine nucleoside, has shown potential therapeutic effects in the treatment of depression. It acts as a neuroprotective agent by promoting axonal regeneration and enhancing neuronal survival. Studies have indicated that inosine can 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. 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. By uncoupling SERT from nNOS, increasing DRN membrane SERT, reducing intercellular serotonin, and lowering negative feedback, serotonergic neuron firing is stimulated, leading to a rapid increase in serotonin concentration in the postsynaptic synaptic cleft, thereby exerting a rapid antidepressant effect independent of serotonin autoreceptor desensitization. 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[2].

We identified the sequence of the key gene gsk in the synthesis of inosine, and designed both upstream and downstream primers for gsk. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the pSIP403-P9-gsk plasmid in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce inosine. Moreover, we also add the kill switch mentioned above into the vector for biosafety.

Build:

We identified the sequence of the key gene gsk in the synthesis of inosine, and designed both upstream and downstream primers for gsk. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the pSIP403-P9-gsk plasmid in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce inosine. Moreover, we also add the kill switch mentioned above into the vector for biosafety. (See more in design safety-suicide switch).

Regrettably, we encountered difficulties when attempting to concatenate the suicide switch with the target gene gsk due to time constraints, which prevented us from successfully constructing the plasmid and transforming it into Lactobacillus plantarum. Consequently, in the subsequent experimental validation, we utilized the plasmid pSIP403-P9-gsk, which expresses only the target gene.

TEST:

We successfully transformed the plasmid into Lactobacillus plantarum L168 , and validation through PCR and agarose gel electrophoresis confirmed the success of this step. Following this, we cultured Lactobacillus plantarum L168 and extracted RNA, with the supernatant being used for metabolite measurement.

Next, we conducted reverse transcription and quantitative polymerase chain reaction (qPCR) experiments to quantify the gene expression levels in Lactobacillus plantarum L168 , both with and without the pSIP403-P9-gsk plasmid. The qPCR results, as shown in figure 3, indicated our designed plasmid effectively and significantly upregulated the gene expression of inosine in Lactobacillus plantarum L168 (Fig. 3).

Figure 3. Relative Expression Analysis of gene gsk by qPCR.

Liquid Chromatograph Mass Spectrometer (LC-MS) was also utilized to examine the production of inosine in the supernatant of Lactobacillus plantarum L168 . It is evident that the level of inosine rose significantly in Lactobacillus plantarum L168 containing the pSIP403-P9-gsk plasmid compared to the control group, revealing that, as anticipated, the gsk gene was effectively expressed in Lactobacillus plantarum L168 and led to inosine production (Fig. 4). You can find more information on the Results page.

Figure 4. Relative levels of inosine between two groups by LC-MS.

Learn:

Neuroprotective Properties of Inosine: Inosine's function as a neuroprotective agent is underscored by its capacity to stimulate axonal regeneration and bolster neuronal survival. This highlights the significance of neurotrophic factors in the maintenance and recuperation of neural health, which is essential for emotional well-being.

Modulation of Neurotransmitter Levels: The research indicates that inosine has the potential to alleviate depressive symptoms by modulating neurotransmitter levels. This suggests that the equilibrium of neurotransmitters plays a pivotal role in the pathophysiology of mood disorders, and interventions that target these levels may offer therapeutic advantages.

Molecular Docking and Interaction Experiments: The study reveals that inosine can bind to the neuronal nitric oxide synthase (nNOS) receptor, a novel discovery that could pave the way for more precise treatments with a reduced incidence of side effects. This approach exemplifies the utility of molecular biology in the identification and validation of new therapeutic targets.

Gut-Brain Axis and Depression: The focus on the gut-brain axis and the exploitation of small molecules produced by the gut microbiota to target the brain presents a promising strategy for developing treatments that can overcome the blood-brain barrier. This approach could lead to the development of more efficacious and rapidly acting antidepressants.

Experimental Validation: The successful transformation of the plasmid into Lactobacillus plantarum L168 and the subsequent validation through PCR and agarose gel electrophoresis, along with the quantification of gene expression and inosine production, establish a robust experimental framework. These methodologies confirm the operability of the engineered system and the generation of the therapeutic molecule of interest.

Future Directions: The study's promising outcomes also underscore the necessity for further research. This includes the exploration of the long-term effects of inosine on neural health, the refinement of delivery methods for molecules produced by the gut microbiota, and the exploration of potential synergies with existing treatment modalities.

In future experiments, we will strive to validate the functionality of the hypoxia-inducible suicide switch and continue to explore the protocols for constructing engineered Lactobacillus plantarum L168 strains, endeavoring to successfully integrate the target gene with the suicide switch.

Design:

L-theanine is a unique amino acid found in tea leaves, constituting 1-2% of their dry weight. It is known for its various health benefits, including the improvement of learning and memory, relaxation, anti-cancer properties, and neuroprotection. Chemically similar to glutamate, L-theanine acts as a competitive antagonist at glutamate receptors such as NMDAR and AMPAR, albeit weakly, thus inhibiting neuronal death. It also regulates the glutamate-glutamine cycle by inhibiting glutamine transport and promoting the expression of the glutamine transporter protein slc38a1, providing neuroprotection against neurodegenerative diseases[1].

L-theanine degrades into glutamate in normal cells, subsequently increasing intracellular concentrations of glutamine and glutathione. Glutathione is a crucial antioxidant, reducing oxidative damage, maintaining striatal neurotransmitter homeostasis, and inhibiting nitric oxide production. In the context of Alzheimer's disease (AD), L-theanine has been shown to alleviate cognitive dysfunction and neurotoxicity induced by beta-amyloid by reducing oxidative damage and inactivating the ERK/p38 kinase and NF-κB pathways[2].

Furthermore, L-theanine also demonstrates potential effects in the treatment of depression. It may improve mood and sleep by increasing serotonin, dopamine, and GABA levels in the brain, which are closely related to mood regulation. L-theanine may also reduce excitotoxicity caused by excess glutamate by its antagonistic action on glutamate receptors, which could be particularly beneficial for patients with depression.

In summary, L-theanine, as a small molecule, shows potential not only in neuroprotection and the treatment of AD but also as a promising candidate for the treatment of depression and other neurological disorders.

We identified the sequences of the key genes glnA of L-theanine, and designed both upstream and downstream primers for them. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the plasmid in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce theanine.

Build:

We identified the sequences of the key genes glnA of L-theanine, and designed both upstream and downstream primers for them. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the plasmid in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce theanine.

TEST:

We successfully transformed the plasmid into Lactobacillus plantarum L168 , and validation through PCR and agarose gel electrophoresis confirmed this achievement. Following this, we cultured Lactobacillus plantarum L168 and conducted RNA extraction, while the supernatant was used for metabolite analysis. We then performed reverse transcription and quantitative polymerase chain reaction (qPCR) to quantify the gene expression levels in Lactobacillus plantarum L168 , both with and without the recombinant plasmid. The qPCR results imply that our designed plasmid significantly upregulated the expression of key genes for theanine in Lactobacillus plantarum L168 compared to the control group (Fig. 5). You can find more information on the Results page.

Figure 5. Relative Expression Analysis of genes glnA by qPCR.

Learn:

We successfully employed the glnA gene to produce theanine within Lactobacillus plantarum L168 . Utilizing PCR and agarose gel electrophoresis techniques, the team confirmed the successful transformation of the target gene within Lactobacillus plantarum L168 . Furthermore, through the application of quantitative polymerase chain reaction (qPCR) technology, we quantified the gene expression levels, demonstrating that the designed plasmid significantly enhanced the expression of key genes associated with theanine. By producing theanine within Lactobacillus plantarum L168 , this paves the way for potential clinical applications, particularly in the development of novel therapeutics for Alzheimer's Disease (AD) and depression.

Design:

Niacin, also known as vitamin B3, is an essential nutrient that offers various health benefits, including lowering cholesterol, alleviating arthritis, and enhancing brain function. In the body, niacin is converted into niacinamide, which forms part of coenzyme I and coenzyme II. These coenzymes are involved in tissue respiration, the oxidation process, and the anaerobic breakdown of carbohydrates. Niacinamide inhibits the formation of melanin, prevents rough skin, and aids in the repair of damaged cells. It is also indispensable for the synthesis of sex hormones.

Recent studies have highlighted the potential of niacin, also known as vitamin B3, in treating Alzheimer's Disease (AD) and depression. Researchers from the Indiana University School of Medicine explored how niacin modulates microglial responses to amyloid plaques in animal models of AD. They discovered that niacin, when administered in laboratory models, restricted the progression of AD. This finding identifies a potential new therapeutic target for AD that can be regulated through the FDA-approved drug Niaspan.

We identified the sequences of the key genes pncA of Niacin,, and designed both upstream and downstream primers for them. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the plasmid in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce niacin.

Build:

We identified the sequences of the key genes pncA of Niacin,, and designed both upstream and downstream primers for them. The selected vector for this purpose was pSIP403. Subsequently, we designed and constructed the plasmid in the DH5α prior to introducing it into the sensory Lactobacillus plantarum L168 to produce niacin.

TEST:

We successfully transformed the plasmid into Lactobacillus plantarum L168 , and validation through PCR and agarose gel electrophoresis confirmed this achievement. Following this, we cultured Lactobacillus plantarum L168 and conducted RNA extraction, while the supernatant was used for metabolite analysis. We then performed reverse transcription and quantitative polymerase chain reaction (qPCR) to quantify the gene expression levels in Lactobacillus plantarum L168 , both with and without the recombinant plasmid. The qPCR results imply that our designed plasmid significantly upregulated the expression of key genes for niacin in Lactobacillus plantarum L168 compared to the control group (Fig. 6). You can find more information on the Results page.

Figure 6. Relative Expression Analysis of genes pncA by qPCR.

Learn:

We successfully employed the pncA gene to produce niacin within Lactobacillus plantarum L168 . Utilizing PCR and agarose gel electrophoresis techniques, the team confirmed the successful transformation of the target gene within Lactobacillus plantarum L168 . Furthermore, through the application of quantitative polymerase chain reaction (qPCR) technology, the team quantified the gene expression levels, demonstrating that the designed plasmid significantly enhanced the expression of key genes associated with niacin. By producing niacin within Lactobacillus plantarum L168 , this study paves the way for potential clinical applications, particularly in the development of novel therapeutics for Alzheimer's Disease (AD) and depression.