BACKGROUND: Alzheimer's disease (AD) is the most common type of neurodegenerative dementia affecting people in their later years of life. The AD prevalence rate has significantly increased due to a lack of early detection technology and low therapeutic efficacy. Despite recent scientific advances, some aspects of AD pathological targets still require special attention. Certain traditionally consumed phytocompounds have been used for thousands of years to treat such pathologies. The standard extract of Gingko biloba (EGB761) is a combination of 13 macro phyto-compounds and various other micro phytocompounds that have shown greater therapeutic potential against the pathology of AD. OBJECTIVE: Strong physiological evidence of cognitive health preservation has been observed in elderly people who keep an active lifestyle. According to some theories, consuming certain medicinal extracts helps build cognitive reserve. We outline the research employing EGB761 as a dual target for AD. METHODS: This study investigates various inhibitory targets against AD using computational approaches such as molecular docking, network pharmacology, ADMET (full form), and bioactivity prediction of the selected compounds. RESULTS: After interaction studies were done for all the phytoconstituents of EGB761, it was concluded that all four of the phytocompounds (kaempferol, isorhamnetin, quercetin, and ginkgotoxin) showed the maximum inhibitory activity against acetylcholinesterase (AChE) and GSK3beta. CONCLUSION: The highly active phytocompounds of EGB761, especially quercetin, kaempferol, and isorhamnetin, have better activity against AChE and GSK3beta than its reported synthetic drug, according to molecular docking and network pharmacology research. These compounds may act on multiple targets in the protein network of AD. The AChE theory was primarily responsible for EGB761's therapeutic efficacy in treating AD.
Title: An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons Cast TP, Boesch DJ, Smyth K, Shaw AE, Ghebrial M, Chanda S Ref: Journal of Neuroscience, 41:392, 2021 : PubMed
Neuroligins (NLGNs) are a class of postsynaptic cell adhesion molecules that interact with presynaptic neurexins (NRXNs) and regulate synapse function. NLGN4 is a member of the NLGN family and consists of a unique amino acid sequence in humans that is not evolutionarily well conserved in rodents. The human-specific NLGN4 gene has been reported to be mutated in many patients with autism and other neurodevelopmental disorders. However, it remained unclear how these mutations might alter the molecular properties of NLGN4 and affect synaptic transmission in human neurons. Here, we describe a severely autistic male patient carrying a single amino acid substitution (R101Q) in the NLGN4 gene. When expressed in HEK293 cells, the R101Q mutation in NLGN4 did not affect its binding affinity for NRXNs or its capacity to form homodimers. This mutation, however, impaired the maturation of NLGN4 protein by inhibiting N-linked glycosylation at an adjacent residue (N102), which is conserved in all NLGNs. As a result, the R101Q substitution significantly decreased the surface trafficking of NLGN4 and increased its retention in the endoplasmic reticulum and Golgi apparatus. In human neurons derived from male stem cell lines, the R101Q mutation also similarly reduced the synaptic localization of NLGN4, resulting in a loss-of-function phenotype. This mutation-induced trafficking defect substantially diminished the ability of NLGN4 to form excitatory synapses and modulate their functional properties. Viewed together, our findings suggest that the R101Q mutation is pathogenic for NLGN4 and can lead to synaptic dysfunction in autism.
The autism-associated synaptic-adhesion gene Neuroligin-4 (NLGN4) is poorly conserved evolutionarily, limiting conclusions from Nlgn4 mouse models for human cells. Here, we show that the cellular and subcellular expression of human and murine Neuroligin-4 differ, with human Neuroligin-4 primarily expressed in cerebral cortex and localized to excitatory synapses. Overexpression of NLGN4 in human embryonic stem cell-derived neurons resulted in an increase in excitatory synapse numbers but a remarkable decrease in synaptic strength. Human neurons carrying the syndromic autism mutation NLGN4-R704C also formed more excitatory synapses but with increased functional synaptic transmission due to a postsynaptic mechanism, while genetic loss of NLGN4 did not significantly affect synapses in the human neurons analyzed. Thus, the NLGN4-R704C mutation represents a change-of-function mutation. Our work reveals contrasting roles of NLGN4 in human and mouse neurons, suggesting that human evolution has impacted even fundamental cell biological processes generally assumed to be highly conserved.
Title: Unique versus Redundant Functions of Neuroligin Genes in Shaping Excitatory and Inhibitory Synapse Properties Chanda S, Hale WD, Zhang B, Wernig M, Sudhof TC Ref: Journal of Neuroscience, 37:6816, 2017 : PubMed
Neuroligins are evolutionarily conserved postsynaptic cell adhesion molecules that interact with presynaptic neurexins. Neurons express multiple neuroligin isoforms that are targeted to specific synapses, but their synaptic functions and mechanistic redundancy are not completely understood. Overexpression or RNAi-mediated knockdown of neuroligins, respectively, causes a dramatic increase or decrease in synapse density, whereas genetic deletions of neuroligins impair synapse function with only minor effects on synapse numbers, raising fundamental questions about the overall physiological role of neuroligins. Here, we have systematically analyzed the effects of conditional genetic deletions of all major neuroligin isoforms (i.e., NL1, NL2, and NL3), either individually or in combinations, in cultured mouse hippocampal and cortical neurons. We found that conditional genetic deletions of neuroligins caused no change or only a small change in synapses numbers, but strongly impaired synapse function. This impairment was isoform specific, suggesting that neuroligins are not functionally redundant. Sparse neuroligin deletions produced phenotypes comparable to those of global deletions, indicating that neuroligins function in a cell-autonomous manner. Mechanistically, neuroligin deletions decreased the synaptic levels of neurotransmitter receptors and had no effect on presynaptic release probabilities. Overexpression of neuroligin-1 in control or neuroligin-deficient neurons increased synaptic transmission and synapse density but not spine numbers, suggesting that these effects reflect a gain-of-function mechanism; whereas overexpression of neuroligin-3, which, like neuroligin-1 is also targeted to excitatory synapses, had no comparable effect. Our data demonstrate that neuroligins are required for the physiological organization of neurotransmitter receptors in postsynaptic specializations and suggest that they do not play a major role in synapse formation.SIGNIFICANCE STATEMENT Human neuroligin genes have been associated with autism, but the cellular functions of different neuroligins and their molecular mechanisms remain incompletely understood. Here, we performed comparative analyses in cultured mouse neurons of all major neuroligin isoforms, either individually or in combinations, using conditional knockouts. We found that neuroligin deletions did not affect synapse numbers but differentially impaired excitatory or inhibitory synaptic functions in an isoform-specific manner. These impairments were due, at least in part, to a decrease in synaptic distribution of neurotransmitter receptors upon deletion of neuroligins. Conversely, the overexpression of neuroligin-1 increased synapse numbers but not spine numbers. Our results suggest that various neuroligin isoforms perform unique postsynaptic functions in organizing synapses but are not essential for synapse formation or maintenance.
Title: Pathogenic mechanism of an autism-associated neuroligin mutation involves altered AMPA-receptor trafficking Chanda S, Aoto J, Lee SJ, Wernig M, Sudhof TC Ref: Mol Psychiatry, 21:169, 2016 : PubMed
Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Although the general synaptic role of neuroligins is undisputed, their specific functions at a synapse remain unclear, even controversial. Moreover, many neuroligin gene mutations were associated with autism, but the pathophysiological relevance of these mutations is often unknown, and their mechanisms of action uninvestigated. Here, we examine the synaptic effects of an autism-associated neuroligin-4 substitution (called R704C), which mutates a cytoplasmic arginine residue that is conserved in all neuroligins. We show that the R704C mutation, when introduced into neuroligin-3, enhances the interaction between neuroligin-3 and AMPA receptors, increases AMPA-receptor internalization and decreases postsynaptic AMPA-receptor levels. When introduced into neuroligin-4, conversely, the R704C mutation unexpectedly elevated AMPA-receptor-mediated synaptic responses. These results suggest a general functional link between neuroligins and AMPA receptors, indicate that both neuroligin-3 and -4 act at excitatory synapses but perform surprisingly distinct functions, and demonstrate that the R704C mutation significantly impairs the normal function of neuroligin-4, thereby validating its pathogenicity.
Title: Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation Chanda S, Marro S, Wernig M, Sudhof TC Ref: Proc Natl Acad Sci U S A, 110:16622, 2013 : PubMed
Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor- or in GABAA receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling.
Title: Exposure to nerve agents: From status epilepticus to neuroinflammation, brain damage, neurogenesis and epilepsy de Araujo Furtado M, Rossetti F, Chanda S, Yourick D Ref: Neurotoxicology, 33:1476, 2012 : PubMed
Epilepsy is a common neurological disorder characterized by an initial injury due to stroke, traumatic brain injury, brain infection, or febrile seizures causing status epilepticus (SE). This phenomenon precedes recurrent (secondary) seizures, the latent period (period without seizures) and downstream appearance of spontaneous recurrent seizures (SRS). Epilepsy inducers include the organophosphorous (OP) compounds modified as chemical warfare nerve agents, such as soman. SE induced by soman is a result of cholinergic system hyperactivity caused by the irreversible inhibition of acetylcholinesterase, and the subsequent increase in the amount of the neurotransmitter acetylcholine at central and peripheral sites. SE leads to profound, permanent, complex and widespread brain damage and associated cognitive and behavioral deficits, accompanied by impaired neurogenesis. Several anticonvulsant and neuroprotective strategies have been studied in order to avoid the epileptogenesis which occurs after SE caused by soman exposure. In recent studies, we showed that SRS occur post-soman exposure and neuropathology can be reduced with diazepam (DZP) and valproic acid (VPA) when administered in combination treatment. These effects are accompanied by neurogenesis seen 15 days post-exposure in the hippocampal dentate gyrus (DG). This review discusses several findings about epilepsy induced by soman exposure such as behavioral changes, EEG anomalies, neuropathology, neuroinflammation, neurogenesis, possible circuitry changes and current strategies for treatment. The soman seizure model is an important model of temporal lobe epilepsy (TLE) and comparable in certain respects with well studied models in the literature such as pilocarpine and kainic acid. All these models together allow for a greater understanding of the different mechanisms of seizure induction, propagation and options for treatment. These studies are very necessary for current military and civilian treatment regimens, against OP nerve agent exposure, which fail to prevent SE resulting in severe neuropathology and epilepsy.
Title: Gastrointestinal acetylcholinesterase activity following endotracheal microinstillation inhalation exposure to sarin in guinea pigs Chanda S, Song J, Rezk P, Sabnekar P, Doctor BP, Sciuto AM, Nambiar MP Ref: Chemico-Biological Interactions, 187:309, 2010 : PubMed
The goal of this study was to assess acetylcholinesterase (AChE) inhibition at different regions of the gastrointestinal (GI) tract following inhalation exposure to nerve agent sarin. Seven major regions of the GI tract were removed from saline control animals (n=3) and 677.4 mg/m(3) sarin-exposed animals at 4h (n=4) and 24h (n=4) post-exposure. AChE activity was determined in blood and homogenized tissue supernatant by specific Ellman's assay using Iso-OMPA, a BChE inhibitor, and expressed as activity/optical density of hemoglobin for blood and activity/mg protein for tissues. Our data showed that the AChE activity was significantly decreased for groups both 4h and 24h post-sarin exposure. Among the seven chosen regions of the guinea pig GI tract, duodenum showed the highest AChE activity in control animals. The AChE activity was significantly decreased in the stomach (p=0.03), duodenum (p=0.029), jejunum (p=0.006), and ileum (p=0.006) 4h following sarin exposure. At 24h post-sarin exposure the AChE activity of duodenum (p=0.029) and ileum (p=0.006) was significantly inhibited. Esophagus showed no inhibition following sarin exposure at both 4h and 24h groups. These results suggest that the AChE activity is different in different regions of the GI tract and highest levels of AChE inhibition following sarin exposure were seen in regions exhibiting higher overall AChE activity and cholinergic function.
Respiratory toxicity and lung injury following inhalation exposure to chemical warfare nerve agent soman was examined in guinea pigs without therapeutics to improve survival. A microinstillation inhalation exposure technique that aerosolizes the agent in the trachea was used to administer soman to anesthetized age and weight matched male guinea pigs. Animals were exposed to 280, 561, 841, and 1121 mg/m(3) concentrations of soman for 4 min. Survival data showed that all saline controls and animals exposed to 280 and 561 mg/m(3) soman survived, while animals exposed to 841, and 1121 mg/m(3) resulted in 38% and 13% survival, respectively. The microinstillation inhalation exposure LCt(50) for soman determined by probit analysis was 827.2mg/m(3). A majority of the animals that died at 1121 mg/m(3) developed seizures and died within 15-30 min post-exposure. There was a dose-dependent decrease in pulse rate and blood oxygen saturation of animals exposed to soman at 5-6.5 min post-exposure. Body weight loss increased with the dose of soman exposure. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase and butyrylcholinesterase activity was inhibited dose-dependently in soman treated groups at 24h. BAL cells showed a dose-dependent increase in cell death and total cell counts following soman exposure. Edema by wet/dry weight ratio of the accessory lung lobe and trachea was increased slightly in soman exposed animals. An increase in total bronchoalveolar lavage fluid protein was observed in soman exposed animals at all doses. Differential cell counts of BAL and blood showed an increase in total lymphocyte counts and percentage of neutrophils. These results indicate that microinstillation inhalation exposure to soman causes respiratory toxicity and acute lung injury in guinea pigs.
