A novel bHLH gene responsive to low nitrogen positively regulates the biosynthesis of medicinal tropane alkaloids in Atropa belladonna.

Medicinal tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are essential anticholinergic drugs specifically produced in several solanaceous plants. Atropa belladonna is one of the most important medicinal plants that produces TAs. Therefore, it is necessary to cultivate new A. belladonna germplasm with the high content of TAs. Here, we found that the levels of TAs were elevated under low nitrogen (LN) condition, and identified a LN-responsive bHLH transcription factor (TF) of A. belladonna (named LNIR) regulating the biosynthesis of TAs. The expression level of LNIR was highest in secondary roots where TAs are synthesized specifically, and was significantly induced by LN. Further research revealed that LNIR directly activated the transcription of hyoscyamine 6ß-hydroxylase gene (H6H) by binding to its promoter, which converts hyoscyamine into anisodamine and subsequently epoxidizes anisodamine to form scopolamine. Overexpression of LNIR upregulated the expression levels of TA biosynthesis genes and consequently led to the increased production of TAs. In summary, we functionally identified a LN-responsive bHLH gene that facilitated the development of A. belladonna with high-yield TAs under the decreased usage of nitrogen fertilizer.

Nelumbo nucifera leaves extract ameliorated scopolamine-induced cognition impairment via enhanced adult hippocampus neurogenesis.

In this presentation, we explored the molecular mechanisms of N. nucifera leaf water extracts (NLWEs) and polyphenol extract (NLPE) on scopolamine-induced cell apoptosis and cognition defects. The administration of NLWE and NLPE did not alter the body weight and serum biomarker rs and significantly ameliorated scopolamine-induced cognition impairment according to Y-maze test analysis. In mice, treatment with scopolamine disrupted normal histoarchitecture in the hippocampus, whereas the administration of NLWE and NLPE reversed the phenomenon. Western blot analysis revealed that scopolamine mitigated the expression of doublecortin (DCX), nestin, and NeuN, and cotreatment with NLWE or NLPE significantly recovered the expression of these proteins. NLWE and NLPE upregulated DCX and NeuN expression in the hippocampus region, as evidenced by immunohistochemical staining analysis of scopolamine-treated mice. NLWE and NLPE obviously elevated brain-derived neurotrophic factor (BDNF) and enhanced its downstream proteins activity. NLWE and NLPE attenuated scopolamine-induced apoptosis by reducing Bax and increased Bcl-2 expression. In addition, scopolamine also triggered apoptosis in human neuroblastoma SH-SY5Y cells whereas co-treatment with NLWE or quercetin-3-glucuronide (Q3G) reversed the phenomenon. NLWE or Q3G enhanced Bcl-2 and reduced Bax expression in the presence of scopolamine in SH-SY5Y cells. NLWE or Q3G recovered the inhibitory effects of scopolamine on neurogenesis and BDNF signals in SH-SY5Y cells. Overall, our results revealed that N. nucifera leaf extracts and Q3G promoted adult hippocampus neurogenesis and prevented apoptosis to mitigate scopolamine-induced cognition dysfunction through the regulation of BDNF signaling pathway.

Orexin-A attenuated motion sickness through modulating neural activity in hypothalamus nuclei.

BACKGROUND AND PURPOSE: We evaluated the hypothesis that central orexin application could counteract motion sickness responses through regulating neural activity in target brain areas. EXPERIMENTAL APPROACH: Thec effects of intracerebroventricular (i.c.v.) injection of orexin-A and SB-334867 (OX1 antagonist) on motion sickness-induced anorexia, nausea-like behaviour (conditioned gaping), hypoactivity and hypothermia were investigated in rats subjected to Ferris wheel-like rotation. Orexin-A responsive brain areas were identified using Fos immunolabelling and were verified via motion sickness responses after intranucleus injection of orexin-A, SB-334867 and TCS-OX2-29 (OX2 antagonist). The efficacy of intranasal application of orexin-A versus scopolamine on motion sickness symptoms in cats was also investigated. KEY RESULTS: Orexin-A (i.c.v.) dose-dependently attenuated motion sickness-related behavioural responses and hypothermia. Fos expression was inhibited in the ventral part of the dorsomedial hypothalamus (DMV) and the paraventricular nucleus (PVN), but was enhanced in the ventral part of the premammillary nucleus ventral part (PMV) by orexin-A (20 µg) in rotated animals. Motion sickness responses were differentially inhibited by orexin-A injection into the DMV (anorexia and hypoactivity), the PVN (conditioned gaping) and the PMV (hypothermia). SB-334867 and TCS-OX2-29 (i.c.v. and intranucleus injection) inhibited behavioural and thermal effects of orexin-A. Orexin-A (60 µg·kg-1) and scopolamine inhibited rotation-induced emesis and non-retching/vomiting symptoms, while orexin-A also attenuated anorexia with mild salivation in motion sickness cats. CONCLUSION AND IMPLICATIONS: Orexin-A might relieve motion sickness through acting on OX1 and OX2 receptors in various hypothalamus nuclei. Intranasal orexin-A could be a potential strategy against motion sickness.

Multiple independent losses of the biosynthetic pathway for two tropane alkaloids in the Solanaceae family.

Hyoscyamine and scopolamine (HS), two valuable tropane alkaloids of significant medicinal importance, are found in multiple distantly related lineages within the Solanaceae family. Here we sequence the genomes of three representative species that produce HS from these lineages, and one species that does not produce HS. Our analysis reveals a shared biosynthetic pathway responsible for HS production in the three HS-producing species. We observe a high level of gene collinearity related to HS synthesis across the family in both types of species. By introducing gain-of-function and loss-of-function mutations at key sites, we confirm the reduced/lost or re-activated functions of critical genes involved in HS synthesis in both types of species, respectively. These findings indicate independent and repeated losses of the HS biosynthesis pathway since its origin in the ancestral lineage. Our results hold promise for potential future applications in the artificial engineering of HS biosynthesis in Solanaceae crops.

Zinc Ortho Methyl Carbonodithioate Improved Pre and Post-Synapse Memory Impairment via SIRT1/p-JNK Pathway against Scopolamine in Adult Mice.

Alzheimer's disease (AD) is globally recognized as a prominent cause of dementia for which efficient treatment is still lacking. New candidate compounds that are biologically potent are regularly tested. We, therefore, hypothesized to study the neuroprotective potential of Zinc Ortho Methyl Carbonodithioate (thereafter called ZOMEC) against Scopolamine (SCOP) induced Alzheimer's disease (AD) model using adult albino mice. We post-administered ZOMEC (30 mg/Kg) into two group of mice for three weeks on daily basis that received either 0.9% saline or SCOP (1 mg/Kg) for initial two weeks. The other two groups of mice received 0.9% saline and SCOP (1 mg/Kg) respectively. After memory related behavioral analysis the brain homogenates were evaluated for the antioxidant potential of ZOMEC and multiple protein markers were examined through western blotting. Our results provide enough evidences that ZOMEC decrease oxidative stress by increasing catalase (CAT) and glutathione S transferase (GST) and decreasing the lipid peroxidation (LPO). The SIRT1 and pre and post synaptic marker proteins, synaptophysin (SYP) as well as post synaptic density protein (PSD-95) expression were also enhanced upon ZOMEC treatment. Furthermore, memory impairment was rescued and ZOMEC appreciably abrogated the Aß accumulation, BACE1 expression C and the p-JNK pathway. The inflammatory protein markers, NF-kß and IL-1ß in ZOMEC treated mice were also comparable with control group. The predicted interaction of ZOMEC with SIRT1 was further confirmed by molecular docking. These findings thus provide initial reports on efficacy of ZOMEC in SCOP induced AD model.

Umbelliferone Ameliorates Memory Impairment and Enhances Hippocampal Synaptic Plasticity in Scopolamine-Induced Rat Model.

Alzheimer's disease (AD) is a neurodegenerative disorder, characterized by memory loss and cognitive decline. Among the suggested pathogenic mechanisms of AD, the cholinergic hypothesis proposes that AD symptoms are a result of reduced synthesis of acetylcholine (ACh). A non-selective antagonist of the muscarinic ACh receptor, scopolamine (SCOP) induced cognitive impairment in rodents. Umbelliferone (UMB) is a Apiaceae-family-derived 7-hydeoxycoumarin known for its antioxidant, anti-tumor, anticancer, anti-inflammatory, antibacterial, antimicrobial, and antidiabetic properties. However, the effects of UMB on the electrophysiological and ultrastructure morphological aspects of learning and memory are still not well-established. Thus, we investigated the effect of UMB treatment on cognitive behaviors and used organotypic hippocampal slice cultures for long-term potentiation (LTP) and the hippocampal synaptic ultrastructure. A hippocampal tissue analysis revealed that UMB attenuated a SCOP-induced blockade of field excitatory post-synaptic potential (fEPSP) activity and ameliorated the impairment of LTP by the NMDA and AMPA receptor antagonists. UMB also enhanced the hippocampal synaptic vesicle density on the synaptic ultrastructure. Furthermore, behavioral tests on male SD rats (7-8 weeks old) using the Y-maze test, passive avoidance test (PA), and Morris water maze test (MWM) showed that UMB recovered learning and memory deficits by SCOP. These cognitive improvements were in association with the enhanced expression of BDNF, TrkB, and the pCREB/CREB ratio and the suppression of acetylcholinesterase activity. The current findings indicate that UMB may be an effective neuroprotective reagent applicable for improving learning and memory against AD.

Recycling of hyoscyamine 6ß-hydroxylase for the in vitro production of anisodamine and scopolamine.

The tropane alkaloids hyoscyamine, anisodamine, and scopolamine are extensively used medicines. In particular, scopolamine has the greatest value in the market. Hence, strategies to enhance its production have been explored as an alternative to traditional field-plant cultivation. In this work, we developed biocatalytic strategies for the transformation of hyoscyamine into its products utilizing a recombinant Hyoscyamine 6ß-hydroxylase (H6H) fusion protein to the chitin-binding domain of the chitinase A1 from Bacillus subtilis (ChBD-H6H). Catalysis was carried out in batch, and recycling of H6H constructions was performed via affinity-immobilization, glutaraldehyde crosslinking, and adsorption-desorption of the enzyme to different chitin matrices. ChBD-H6H utilized as free enzyme achieved complete conversion of hyoscyamine in 3- and 22-h bioprocesses. Chitin particles demonstrated to be the most convenient support for ChBD-H6H immobilization and recycling. Affinity-immobilized ChBD-H6H operated in a three-cycle bioprocess (3 h/cycle, 30 °C) yielded in the first and third reaction cycle 49.8% and 22.2% of anisodamine and 0.7% and 0.3% of scopolamine, respectively. However, glutaraldehyde crosslinking decreased enzymatic activity in a broad range of concentrations. Instead, the adsorption-desorption approach equaled the maximal conversion of the free enzyme in the first cycle and retained higher enzymatic activity than the carrier-bound strategy along the consecutive cycles. The adsorption-desorption strategy permitted the reutilization of the enzyme in a simple and economical manner while exploiting the maximal conversion activity displayed by the free enzyme. This approach is valid since other enzymes present in the E. coli lysate do not interfere with the reaction. KEY POINTS: • A biocatalytic system for anisodamine and scopolamine production was developed. • Affinity-immobilized ChBD-H6H in ChP retained catalytic activity. • Enzyme-recycling by adsorption-desorption strategies improves product yields.

Revealing evolution of tropane alkaloid biosynthesis by analyzing two genomes in the Solanaceae family.

Tropane alkaloids (TAs) are widely distributed in the Solanaceae, while some important medicinal tropane alkaloids (mTAs), such as hyoscyamine and scopolamine, are restricted to certain species/tribes in this family. Little is known about the genomic basis and evolution of TAs biosynthesis and specialization in the Solanaceae. Here, we present chromosome-level genomes of two representative mTAs-producing species: Atropa belladonna and Datura stramonium. Our results reveal that the two species employ a conserved biosynthetic pathway to produce mTAs despite being distantly related within the nightshade family. A conserved gene cluster combined with gene duplication underlies the wide distribution of TAs in this family. We also provide evidence that branching genes leading to mTAs likely have evolved in early ancestral Solanaceae species but have been lost in most of the lineages, with A. belladonna and D. stramonium being exceptions. Furthermore, we identify a cytochrome P450 that modifies hyoscyamine into norhyoscyamine. Our results provide a genomic basis for evolutionary insights into the biosynthesis of TAs in the Solanaceae and will be useful for biotechnological production of mTAs via synthetic biology approaches.

Tear film instability is associated with weakened colocalization between occludin and MUC5AC in scopolamine-induced dry eye disease (DED) rats.

PURPOSE: Dry eye disease (DED) is a disease with tear film instability because of multiple factors. This study was conducted to explore roles of occludin and MUC5AC in tear film instability in DED rat model. METHODS: A total of 20 SD rats were divided into DED group (n = 10) and normal control (NC) group (n = 10). DED rat model was established by subcutaneously injecting with scopolamine hydrobromide. Clinical examinations, including tear breakup time (tBUT), Schirmer's test and corneal fluorescein staining, were conducted to determine corneal functions. Transmission electron microscopy was used to measure the ultrastructures of corneal epithelial cells. Western blotting assay was used to identify occludin expression in corneal tissues of DED rats. Real-time PCR (RT-PCR) was performed to verify gene transcription of occludin and MUC5AC. Colocalization between occludin and MUC5AC was identified with confocal fluorescence microscopy. RESULTS: Tear breakup time was significantly shorter, and corneal fluorescein staining score was predominantly higher in DED rats compared to those in normal rats (P < 0.05). Normal rats showed a steady tear secretion throughout the whole experiments, while DED rats showed a dramatic reduction on day 14. DED rats demonstrated ultrastructural damage of Golgi apparatus and endoplasmic reticulum in corneal epithelial cells. Occludin and MUC5AC expressions were significantly downregulated in corneal tissue of DED rats compared with those of normal rats (P < 0.05). Percentage of occludin-MUC5AC-colocalized corneal epithelial cells in DED rats was significantly less compared with those in normal rats (P < 0.01). CONCLUSIONS: Tear film stability was damaged in scopolamine-induced DED rats because of the weakened colocalization between occludin and MUC5AC molecule. This study would provide a potential clue for the pathogenesis and a promising theoretical basis for clinical work of DED.

Wheatgrass extract imparts neuroprotective actions against scopolamine-induced amnesia in mice.

The rich and diverse phytoconstituents of wheatgrass have established it as a natural antioxidant and detoxifying agent. The anti-inflammatory potential of wheatgrass has been studied extensively. However, the neuroprotective potential of wheatgrass has not been studied in depth. In this study, we investigated the neuroprotective response of wheatgrass against age-related scopolamine-induced amnesia in mice. Scopolamine is an established anticholinergic drug that demonstrates the behavioural and molecular characteristics of Alzheimer's disease. In the current study, wheatgrass extracts (prepared from 5 and 7 day old plantlets) were administered to scopolamine-induced memory deficit mice. The Morris water maze (MWM) and Y-maze tests demonstrated that wheatgrass treatment improves the behavior and simultaneously enhances the memory of amnesic mice. We further evaluated the expression of neuroinflammation related genes and proteins in the hippocampal region of mice. Wheatgrass significantly upregulated the mRNA and protein expression of neuroprotective markers such as BDNF and CREB in scopolamine-induced mice. Simultaneously, wheatgrass also downregulated the expression of inflammatory markers such as TNF-α and tau genes in these mice. The treatment of scopolamine-induced memory impaired mice with wheatgrass resulted in an elevation in the level of the phosphorylated form of ERK and Akt proteins. Wheatgrass treatment of mice also regulated the phosphorylation of tau protein and simultaneously prevented its aggregation in the hippocampal region of the brain. Overall, this study suggests the therapeutic potential of wheatgrass in the treatment of age-related memory impairment, possibly through the involvement of ERK/Akt-CREB-BDNF pathway and concomitantly ameliorating the tau-related pathogenesis.

Insights into the hippocampus proteome and phosphorylation modification alterations in C57BL/6 revealed the memory improvement mechanisms of a walnut-derived peptide.

This work aimed to explore the underlying mechanisms of memory improvement effects of a walnut derived peptide WNP-10. The morris water maze test, combined with ultrastructural observation, hematoxylin and eosin and Nissl staining showed that WNP-10 significantly improved the learning and memory capability of the scopolamine-injured mice. The four-dimensional label-free quantification proteomics analysis identified 88 differentially expressed proteins in the WNP-10-treated group compared with scopolamine-induced impairment group. Pathway enrichment analysis and western blotting demonstrated that the WNP-10 can regulate the phosphatidylinositol-3-phosphate 5-kinase, cathepsin L, N-acetylgalactosamine 6-sulfate sulfatase and AP-3 complex subunit mu-1 expression to affect inositol phosphate metabolism, thereby maintaining lysosome homeostasis in scopolamine-injured mice. Notably, the results of phosphoproteomics demonstrated that WNP-10 administration resulted in the increased phosphorylation of phosphatidylinositol-3-phosphate 5-kinase. These findings provide novel insights into the underlying mechanism of memory improvement of walnut peptides.

Beneficial Effects of Snail Helix aspersa Extract in an Experimental Model of Alzheimer's Type Dementia.

BACKGROUND: Alzheimer's disease (AD) is a complex neurodegenerative disease with multifactorial etiology, unsatisfactory treatment, and a necessity for broad-spectrum active substances for cure. The mucus from Helix aspersa snail is a mixture of bioactive molecules with antimicrobial, anti-inflammatory, antioxidant, and anti-apoptotic effects. So far there are no data concerning the capacity of snail extract (SE) to affect neurodegenerative disorders. OBJECTIVE: The effects of SE from Helix aspersa on learning and memory deficits in Alzheimer's type dementia (ATD) induced by scopolamine (Sco) in male Wistar rats were examined and some mechanisms of action underlying these effects were evaluated. METHODS: SE (0.5 mL/100 g) was applied orally through a food tube for 16 consecutive days: 5 days before and 11 days simultaneously with Sco (2 mg/kg, intraperitoneally). At the end of Sco treatment, using behavioral methods, we evaluated memory performance. Additionally, in cortex and hippocampus the acetylcholinesterase (AChE) activity, acetylcholine and monoamines (dopamine, noradrenaline, and serotonin) content, levels of main oxidative stress markers, and expression of brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) were determined. RESULTS: We demonstrated that, according to all behavioral tests used, SE significantly improved the cognitive deficits induced by Sco. Furthermore, SE possessed AChE inhibitory activity, moderate antioxidant properties and the ability to modulate monoamines content in two brain structures. Moreover, multiple SE applications not only restored the depressed by Sco expression of CREB and BDNF, but significantly upregulated it. CONCLUSION: Summarizing results, we conclude that complex mechanisms underlie the beneficial effects of SE on impaired memory in Alzheimer's type dementia.

Inhibition of scopolamine-induced memory and mitochondrial impairment by betanin.

Mitochondrial dysfunction and oxidative stress are identified to contribute to the mechanisms responsible for the pathogenesis of Alzheimer's disease (AD). Scopolamine (SCO) as a potent drug for inducing memory and learning impairment is associated with mitochondrial dysfunction and oxidative stress. In AD clinical trials molecules with antioxidant properties have shown modest benefit. Betanin as a multifunctional molecule with powerful antioxidative properties may be effective in the treatment of neurodegenerative. Hence, this study was designed to investigate the possible therapeutic effect of betanin against SCO-induced AD on Wistar rats. SCO (1 mg/kg) was administrated intraperitoneally to induce the AD in Wistar rats. The rats were treated with betanin doses (25 mg/kg and 50 mg/kg) intraperitoneally for 9 consecutive days. At the end of the 9th day, the animals were subjected to behavioral examination such as novel object recognition and passive avoidance tests and killed to study the mitochondrial and histological parameters. The results showed attenuation of SCO-induced memory and learning impairment by betanin at 50 mg/kg dose. Also, mitochondrial toxicity parameters such as mitochondrial membrane potential collapse, mitochondrial swelling, decreased activity of succinate dehydrogenase, and reactive oxygen species (ROS) production were reversed by betanin (50 mg/kg) compared to the SCO group. In addition, the ameliorative effect of betanin against SCO was demonstrated in histopathological results of hippocampus. The present investigation established that the betanin ameliorates the SCO-induced memory impairments, tissue injuries, and mitochondrial dysfunction by reducing mitochondrial ROS, which may be due to the potent antioxidant action of betanin.

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna.

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs' production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Decreased efficacy of the ketamine and scopolamine-induced sustained antidepressant-like effects in rats receiving metformin.

BACKGROUND: Metformin is the most widely used drug for treating type 2 diabetes mellitus (DM), which frequently co-occurs with depressive disorders. Thus, patients with depression are likely to receive metformin. Metformin activates AMP-activated kinase (AMPK), which inhibits mechanistic target of rapamycin complex 1 (mTORC1) signaling. mTORC1 activation is essential for the antidepressant effects of ketamine and scopolamine. Thus, we hypothesized that metformin may attenuate ketamine- or scopolamine-induced antidepressant efficacies by blocking their mTORC1 activation. METHODS: We assessed the acute and sustained antidepressant-like actions of ketamine and scopolamine in male Sprague-Dawley rats subjected to the forced swim test with or without metformin pretreatment. The expressions of AMPK, mTORC1, and brain-derived neurotrophic factor (BDNF) in their prefrontal cortex were assessed. RESULTS: Metformin (50 mg/kg) attenuated the sustained, but not acute, antidepressant-like effects of ketamine (10 mg/kg) and scopolamine (25 µg/kg). Although metformin reduced mTORC1 downstream activated P70S6K, it did not significantly alter mTORser2448 activation and even increased BDNF expression. Notably, ketamine, scopolamine, and metformin all exerted significant antidepressant-like actions, as evidenced by increased AMPK phosphorylation and BDNF expression. CONCLUSIONS: Metformin-induced attenuation of sustained antidepressant-like effects are not directly dependent on AMPK-deactivated mTORC1. Our results indicate the complexity of interactions between AMPK, BDNF, and mTORC1. Further research, including mechanistic studies, is warranted to comprehensively evaluate the application of metformin in patients receiving mTORC1-based antidepressants.

Euonymus alatus Twig Extract Protects against Scopolamine-Induced Changes in Brain and Brain-Derived Cells via Cholinergic and BDNF Pathways.

In the current study, the therapeutic and preventive effects of Euonymus alatus (EA) twig extract were investigated in a mouse model of cognitive deficit and B35 cells. Twig extract 1 was extracted with 70% ethanol and later twig extract 2 was extracted through liquid-liquid extraction with 70% ethanol and hexane. EA twig 2 (300 mg/kg) along with the standard drug donepezil (5 mg/kg) were orally administered to the mice for 34 days. Scopolamine was given intraperitoneally for 7 days. Administration of EA twig extract 2 significantly improved the passive avoidance test (PAT) in mice. EA twigs extract also restored the scopolamine-reduced brain-derived neurotrophic factor (BDNF)/extracellular regulated kinase (ERK)/cyclic AMP responsive element binding protein (CREB) signaling in B35 cells and the mouse hippocampus. In addition, EA twig extract significantly inhibited the acetylcholine esterase (AChE) activity in B35 cells in a dose-dependent manner. Chromatography and ESI MS analysis of EA twig extract revealed the presence of flavonoids; epicatechin, taxifolin, aromadendrin, and naringenin with catechin being the most abundant. These flavonoids exerted protective effects alone and had the possibility of synergistic effects in combination. Our work unmasks the ameliorating effect of EA twig extract 2 on scopolamine-associated cognitive impairments through the restoration of cholinergic systems and the BDNF/ERK/CREB pathway.

Development of Atropa belladonna L. Plants with High-Yield Hyoscyamine and without Its Derivatives Using the CRISPR/Cas9 System.

Atropa belladonna L. is one of the most important herbal plants that produces hyoscyamine or atropine, and it also produces anisodamine and scopolamine. However, the in planta hyoscyamine content is very low, and it is difficult and expensive to independently separate hyoscyamine from the tropane alkaloids in A. belladonna. Therefore, it is vital to develop A. belladonna plants with high yields of hyoscyamine, and without anisodamine and scopolamine. In this study, we generated A. belladonna plants without anisodamine and scopolamine, via the CRISPR/Cas9-based disruption of hyoscyamine 6ß-hydroxylase (AbH6H), for the first time. Hyoscyamine production was significantly elevated, while neither anisodamine nor scopolamine were produced, in the A. belladonna plants with homozygous mutations in AbH6H. In summary, new varieties of A. belladonna with high yields of hyoscyamine and without anisodamine and scopolamine have great potential applicability in producing hyoscyamine at a low cost.

PET Imaging Estimates of Regional Acetylcholine Concentration Variation in Living Human Brain.

Acetylcholine (ACh) has distinct functional roles in striatum compared with cortex, and imbalance between these systems may contribute to neuropsychiatric disease. Preclinical studies indicate markedly higher ACh concentrations in the striatum. The goal of this work was to leverage positron emission tomography (PET) imaging estimates of drug occupancy at cholinergic receptors to explore ACh variation across the human brain, because these measures can be influenced by competition with endogenous neurotransmitter. PET scans were analyzed from healthy human volunteers (n = 4) and nonhuman primates (n = 2) scanned with the M1-selective radiotracer [11C]LSN3172176 in the presence of muscarinic antagonist scopolamine, and human volunteers (n = 10) scanned with the α4ß2* nicotinic ligand (-)-[18F]flubatine during nicotine challenge. In all cases, occupancy estimates within striatal regions were consistently lower (M1/scopolamine human scans, 31 ± 3.4% occupancy in striatum, 43 ± 2.9% in extrastriatal regions, p = 0.0094; nonhuman primate scans, 42 ± 26% vs. 69 ± 28%, p < 0.0001; α4ß2*/nicotine scans, 67 ± 15% vs. 74 ± 16%, p = 0.0065), indicating higher striatal ACh concentration. Subject-level measures of these concentration differences were estimated, and whole-brain images of regional ACh concentration gradients were generated. These results constitute the first in vivo estimates of regional variation in ACh concentration in the living brain and offer a novel experimental method to assess potential ACh imbalances in clinical populations.

Engineered Microbes for Producing Anticholinergics.

The tropane alkaloids (TAs) hyoscyamine and scopolamine function as acetylcholine receptor antagonists and are used clinically as parasympatholytics to treat neuromuscular disorders in humans. While TAs are synthesized in a small subset of plant families, these specialized metabolites are only accumulated in limited quantities in plant organs. The complex chemical structures of these compounds make their industrial production by chemical synthesis very challenging, Therefore, the supply of these TAs still relies on intensive farming of Duboisia shrubs in tropical countries. Many adverse factors such as climate fluctuations and pandemics can thus influence annual world production. Based on the landmark microbial production of the antimalarial semi-synthetic artemisinin, the Smolke group recently developed a yeast cell factory capable of de novo synthesizing hyoscyamine and scopolamine, thus paving the way for an alternative production of these compounds.

AGLPM and QMDDQ peptides exert a synergistic action on memory improvement against scopolamine-induced amnesiac mice.

This study aimed to explore the synergistic action of pentapeptides Gln-Met-Asp-Asp-Gln (QMDDQ) and Ala-Gly-Leu-Pro-Met (AGLPM) on memory improvement against scopolamine-induced impairment in mice compared to those of either peptide alone. In behavioral tests, the codelivery of QMDDQ and AGLPM was superior to the individual supplements of either peptide alone not only in enhancing the memory ability at training trials but also in recovering the memory impairment in scopolamine-induced amnesiac mice in test trials. Furthermore, combination treatment with QMDDQ and AGLPM could significantly reduce the acetylcholinesterase (AChE) level and increase the acetylcholine (ACh) level in the hippocampus, and noticeably improve the pathological morphology of the neuron cells in hippocampal regions CA1 and CA2 and dentate gyrus (DG). The findings indicated that the combination treatment with QMDDQ and AGLPM could improve the memory function by regulating the cholinergic system.