Induction of Gut Microbial Tryptamine by SARS-CoV-2 in Nonhuman Primate Model Consistent with Tryptamine-Induced Model of Neurodegeneration.

The author discussed recently the possible molecular mechanisms that cause the COVID-19 disease symptoms. Here the analysis of the recent experimental data supports the hypothesis that production of the gut microbial tryptamine can be induced by the SARS-CoV-2 fecal viral activity due to the selective pressure or positive selection of tryptamine-producing microorganisms. In this report, the author suggests that the mechanism of microbial selection bases on the abilities of tryptamine to affect the viral nucleic acid. In other words, the gut microorganisms producing tryptamine are more resistant to SARS-CoV-2 fecal viral activity than microorganisms producing no tryptamine. Earlier we demonstrated the induction of neurodegeneration by tryptamine in human cells and mouse brain. Furthermore, we were able to uncover the human gut bacteria associated with Alzheimer's disease (AD) using PCR testing of human fecal samples with the new-designed primers targeting the tryptophan-tryptamine pathway. Likely, SARS-CoV-2 is one of the selective pressure factors in the cascade accelerating the neurodegenerative process in AD. This suggestion is consistent with a higher proportion of AD patients among COVID-19 related victims. Gut microbial tryptamine increase due to the viral infection-induced dysbiosis can synergize and potentiate the tryptamine cytotoxicity, necrotizing ability and other properties as a virulence factor.

Towards Understanding COVID-19: Molecular Insights, Co-infections, Associated Disorders, and Aging.

BACKGROUND: COVID-19 can be related to any diseases caused by microbial infection(s) because 1) co-infection with COVID-19-related virus and other microorganism(s) and 2) because metabolites produced by microorganisms such as bacteria, fungi, and protozoan can be involved in necrotizing pneumonia and other necrotizing medical conditions observed in COVID-19. OBJECTIVE: By way of illustration, the microbial metabolite of aromatic amino acid tryptophan, a biogenic amine tryptamine inducing neurodegeneration in cell and animal models, also induces necrosis. METHODS: This report includes analysis of COVID-19 positivity by zip codes in Florida and relation of the positivity to population density, possible effect of ecological and social factors on spread of COVID-19, autopsy analysis of COVID-19 cases from around the world, serum metabolomics analysis, and evaluation of autoantigenome related to COVID-19. RESULTS: In the present estimations, COVID-19 positivity percent per zip code population varied in Florida from 4.65% to 44.3% (February 2021 data). COVID-19 analysis is partially included in my book Microbial Metabolism and Disease (2021). The autoantigenome related to COVID-19 is characterized by alterations in protein biosynthesis proteins including aminoacyl-tRNA synthetases. Protein biosynthesis alteration is a feature of Alzheimer's disease. Serum metabolomics of COVID-19 positive patients show alteration in shikimate pathway metabolism, which is associated with the presence of Alzheimer's disease-associated human gut bacteria. CONCLUSION: Such alterations in microbial metabolism and protein biosynthesis can lead to toxicity and neurodegeneration as described earlier in my book Protein Biosynthesis Interference in Disease (2020).

Discovery of Gut Bacteria Specific to Alzheimer's Associated Diseases is a Clue to Understanding Disease Etiology: Meta-Analysis of Population-Based Data on Human Gut Metagenomics and Metabolomics.

Alzheimer's disease (AD)-associated sequence (ADAS) of cultured fecal bacteria was discovered in human gut targeted screening. This study provides important information to expand our current understanding of the structure/activity relationship of ADAS and putative inhibitors/activators that are potentially involved in ADAS appearance/disappearance. The NCBI database analysis revealed that ADAS presents at a large proportion in American Indian Oklahoman (C&A) with a high prevalence of obesity/diabetes and in colorectal cancer (CRC) patients from the US and China. An Oklahoman non-native group (NNI) showed no ADAS. Comparison of two large US populations reveals that ADAS is more frequent in individuals aged ≥66 and in females. Prevalence and levels of fecal metabolites are altered in the C&A and CRC groups versus controls. Biogenic amines (histamine, tryptamine, tyramine, phenylethylamine, cadaverine, putrescine, agmatine, spermidine) that present in food and are produced by gut microbiota are significantly higher in C&A (e.g., histamine/histidine 95-fold) versus NNI (histamine/histidine 16-fold). The majority of these bio-amines are cytotoxic at concentrations found in food. Inositol phosphate signaling implicated in AD is altered in C&A and CRC. Tryptamine stimulated accumulation of inositol phosphate. The seizure-eliciting tryptamine induced cytoplasmic vacuolization and vesiculation with cell fragmentation. Present additions of ADAS-carriers at different ages including infants led to an ADAS-comprising human sample size of 2,830 from 27 studies from four continents (North America, Australia, Asia, Europe). Levels of food-derived monoamine oxidase inhibitors and anti-bacterial compounds, the potential modulators of ADAS-bacteria growth and biogenic amine production, were altered in C&A versus NNI. ADAS is attributable to potentially modifiable risk factors of AD associated diseases.

Diet-Related Metabolic Perturbations of Gut Microbial Shikimate Pathway-Tryptamine-tRNA Aminoacylation-Protein Synthesis in Human Health and Disease.

Human gut bacterial Na(+)-transporting NADH:ubiquinone reductase (NQR) sequence is associated with Alzheimer disease (AD). Here, Alzheimer disease-associated sequence (ADAS) is further characterized in cultured spore-forming Clostridium sp. Tryptophan and NQR substrate ubiquinone have common precursor chorismate in microbial shikimate pathway. Tryptophan-derived tryptamine presents in human diet and gut microbiome. Tryptamine inhibits tryptophanyl-tRNA synthetase (TrpRS) with consequent neurodegeneration in cell and animal models. Tryptophanyl-tRNA synthetase inhibition causes protein biosynthesis impairment similar to that revealed in AD. Tryptamine-induced TrpRS gene-dose reduction is associated with TrpRS protein deficiency and cell death. In animals, tryptamine treatment results in toxicity, weight gain, and prediabetes-related hypoglycemia. Sequence analysis of gut microbiome database reveals 89% to 100% ADAS nucleotide identity in American Indian (Cheyenne and Arapaho [C&A]) Oklahomans, of which ~93% being overweight or obese and 50% self-reporting type 2 diabetes (T2D). Alzheimer disease-associated sequence occurs in 10.8% of C&A vs 1.3% of healthy American population. This observation is of considerable interest because T2D links to AD and obesity. Alzheimer disease-associated sequence prevails in gut microbiome of colorectal cancer, which linked to AD. Metabolomics revealed that tryptamine, chorismate precursor quinate, and chorismate product 4-hydroxybenzoate (ubiquinone precursor) are significantly higher, while tryptophan-containing dipeptides are lower due to tRNA aminoacylation deficiency in C&A compared with non-native Oklahoman who showed no ADAS. Thus, gut microbial tryptamine overproduction correlates with ADAS occurrence. Antibiotic and diet additives induce ADAS and tryptamine. Mitogenic/cytotoxic tryptamine cause microbial and human cell death, gut dysbiosis, and consequent disruption of host-microbe homeostasis. Present analysis of 1246 participants from 17 human gut metagenomics studies revealed ADAS in cell death diseases.

Towards an Integrative Understanding of tRNA Aminoacylation-Diet-Host-Gut Microbiome Interactions in Neurodegeneration.

Transgenic mice used for Alzheimer's disease (AD) preclinical experiments do not recapitulate the human disease. In our models, the dietary tryptophan metabolite tryptamine produced by human gut microbiome induces tryptophanyl-tRNA synthetase (TrpRS) deficiency with consequent neurodegeneration in cells and mice. Dietary supplements, antibiotics and certain drugs increase tryptamine content in vivo. TrpRS catalyzes tryptophan attachment to tRNAtrp at initial step of protein biosynthesis. Tryptamine that easily crosses the blood-brain barrier induces vasculopathies, neurodegeneration and cell death via TrpRS competitive inhibition. TrpRS inhibitor tryptophanol produced by gut microbiome also induces neurodegeneration. TrpRS inhibition by tryptamine and its metabolites preventing tryptophan incorporation into proteins lead to protein biosynthesis impairment. Tryptophan, a least amino acid in food and proteins that cannot be synthesized by humans competes with frequent amino acids for the transport from blood to brain. Tryptophan is a vulnerable amino acid, which can be easily lost to protein biosynthesis. Some proteins marking neurodegenerative pathology, such as tau lack tryptophan. TrpRS exists in cytoplasmic (WARS) and mitochondrial (WARS2) forms. Pathogenic gene variants of both forms cause TrpRS deficiency with consequent intellectual and motor disabilities in humans. The diminished tryptophan-dependent protein biosynthesis in AD patients is a proof of our model-based disease concept.

Geographical Distribution and Diversity of Gut Microbial NADH:Ubiquinone Oxidoreductase Sequence Associated with Alzheimer's Disease.

Earlier we reported induction of neurotoxicity and neurodegeneration by tryptophan metabolites that link the metabolic alterations to Alzheimer's disease (AD). Tryptophan is a product of Shikimate pathway (SP). Human cells lack SP, which is found in human gut bacteria exclusively using SP to produce aromatic amino acids (AAA). This study is a first attempt toward gene-targeted analysis of human gut microbiota in AD fecal samples. The oligonucleotide primers newly-designed for this work target SP-AAA in environmental bacteria associated with human activity. Using polymerase chain reaction (PCR), we found unique gut bacterial sequence in most AD patients (18 of 20), albeit rarely in controls (1 of 13). Cloning and sequencing AD-associated PCR products (ADPP) enables identification of Na(+)-transporting NADH: Ubiquinone reductase (NQR) in Clostridium sp. The ADPP of unrelated AD patients possess near identical sequences. NQR substrate, ubiquinone is a SP product and human neuroprotectant. A deficit in ubiquinone has been determined in a number of neuromuscular and neurodegenerative disorders. Antibacterial therapy prompted an ADPP reduction in an ADPP-positive control person who was later diagnosed with AD-dementia. We explored the gut microbiome databases and uncovered a sequence similarity (up to 97%) between ADPP and some healthy individuals from different geographical locations. Importantly, our main finding of the significant difference in the gut microbial genotypes between the AD and control human populations is a breakthrough.

Tryptamine induces axonopathy and mitochondriopathy mimicking neurodegenerative diseases via tryptophanyl-tRNA deficiency.

Neurodegeneration is induced by tryptamine, a human diet constituent, which easily crosses the blood/brain barrier. Tryptamine neurotoxicity, caused by tryptophanyl-tRNA synthetase (TrpRS) inhibition and downregulation leads to tryptophanyl-tRNA deficiency and synthesis of aberrant proteins. We identified axonal defects in hippocampus of tryptamine- treated mice similar to those observed in human brain of patients with Alzheimer's disease, multiple sclerosis and epilepsy using anti-TrpRS site-directed antibodies. The axonal defects are characterized by swellings that accumulate abnormal amounts of helical filaments and amyloid. Tryptamine produced a decreased density of somatic mitochondria concomitant with neuronal loss in mouse hippocampus. In addition, tryptamine evoked accumulation and clustering of small mitochondria in mouse hippocampus causing axonal swellings. Similarly, mitochondrial fission, fusion and clustering were revealed in human neuronal cells after tryptamine administration. Moreover the tryptamine-induced mitochondrial neuropathology includes electron-dense deposits comprising helical fibrils, cristae disruption, cristolysis, mitochondrial swelling and mitochondria-derived vesicles. TrpRS+ helical filamentous tangles formed in both neuronal and kidney cells following tryptamine treatment suggest a tryptamine broad cytotoxic repertoire in damaging vital organs. Tryptamine elicited vesicularization of inner and outer mitochondrial membranes, axonal and cell membranes. Ultrastructurally, fragmentation of swollen degenerated mitochondria, small mitochondria clustering and neurofibrillary tangles are associated with axonal membrane protrusions attributed as neuritic swellings at a lower magnification. TrpRS+ axonal swellings associated with neuropathology of patients and tryptamine-treated human cells suggest that under toxic concentrations, tryptamine is implicated as a causative agent in neurodegeneration resembling that defining a number of human diseases.

Chaperon-like Activation of Serum-Inducible Tryptophanyl-tRNA Synthetase Phosphorylation through Refolding as a Tool for Analysis of Clinical Samples.

Tryptophanyl-tRNA synthetase (TrpRS) expression alters in colorectal (CRC), pancreatic (PC), and cervical (CC) cancers. Here, phosphorylation of unfolded TrpRS and its fragments is stimulated by human cancer sera (CS; n = 13) and serum of rabbit tumor induced by Rous sarcoma virus, unaffected by donor sera (NS; 11/15) and abolished by alkaline phosphatase. At 20 years of follow-up, serum-inducible TrpRS phosphorylation found years before healthy donors (3/15) diagnosed with PC, CRC, or leukemia. I have examined a specificity of serum-inducible TrpRS phosphorylation and found, surprisingly, that serine phosphorylation of unfolded TrpRS is stimulated by anti-TrpRS rabbit antisera but is unaffected by rabbit nonimmune sera and antisera to other antigens. Anti-TrpRS immunoglobulin G (IgG) inhibits phosphorylation of full-length TrpRS and stimulates phosphorylation of its 20-kDa fragment. Phosphorylation of this fragment is stimulated also by CS but not NS. 2-Mercaptoethanol and cyclic AMP exerted synergistic inhibitory effect on TrpRS phosphorylation. Anti-TrpRS sera and casein act as chaperones increasing TrpRS phosphorylation through refolding. Histone-specific protein kinase activity in CS (n = 44) and anti-TrpRS sera was lower than that in NS (n = 11), rabbit nonimmune sera and antisera to other antigens. TrpRS inhibitors, tryptamine, and tryptophanol stimulate in vivo accumulation of enzymatically inactive, nonphosphorylated, aggregated and anti-TrpRS IgG refoldable TrpRS. Phosphorylation of postsurgical tissues (n = 18) reveals TrpRS in ovarian cancer (OVC) and CC but not in normal placenta and liver. In OVC, TrpRS phosphorylation increase correlates with elevated tryptophan-dependent ATP-inorganic pyrophosphate exchange. Although not inducing cancer, TrpRS triggers signaling concomitant with cancer.

Hypoxia signature of splice forms of tryptophanyl-tRNA synthetase marks pancreatic cancer cells with distinct metastatic abilities.

OBJECTIVES: Pancreatic cancer is one of most deadly because of its aggressive growth and high metastatic ability that correlates with intratumoral hypoxia. Earlier diagnosis and prognosis marker of pancreatic cancer is not yet available. In colorectal cancer, protein biosynthesis enzyme, tryptophanyl-tRNA synthetase (TrpRS), is up-regulated in good-prognosis tumors and down-regulated in metastatic poor-prognosis tumors. Tryptophanyl-tRNA synthetase status in pancreatic cancer is unknown. To correlate metastatic ability with hypoxia and TrpRS as a possible prognostic marker, we examined mRNA and protein expression in 2 human pancreatic cancer cell lines with different metastatic abilities and TrpRS levels using our site-specific monoclonal antibodies directed to conformation-dependent epitopes on pancreatic TrpRS. METHODS: Pancreatic MIAPaCa-2, Panc-1, cervical HeLa, and prostate cancer PC-3 cells were cultivated under normoxia or in hypoxic chamber. Expression of full-length TrpRS, antiangiogenic TrpRS, cyclin B1, hypoxia-inducible factor 1α, and Glut-1 was determined with reverse transcriptase-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS: We demonstrate that hypoxia regulates differentially TrpRS splice forms. Pronounced down-regulation of full-length TrpRS by hypoxia is concomitant with higher metastatic ability. CONCLUSIONS: Tryptophanyl-tRNA synthetase down-regulation by hypoxia may be a factor responsible for low TrpRS in tumors with high metastatic ability. Tryptophanyl-tRNA synthetase recognizability is important for pancreatic cancer prognosis and as a new target for metastasis treatment.

Tryptamine-induced tryptophanyl-tRNAtrp deficiency in neurodifferentiation and neurodegeneration interplay: progenitor activation with neurite growth terminated in Alzheimer's disease neuronal vesicularization and fragmentation.

Tryptophanyl-tRNA synthetase (TrpRS) catalyzes tryptophanyl-tRNAtrp formation. At concentrations exceeding tryptophan, tryptamine inhibits TrpRS. This leads in tryptophanyl-tRNA deficiency and synthesis of aberrant proteins. Tryptamine presents in food and crosses blood-brain barrier. The purpose of this study is to test the hypothesis that tryptamine-induced changes in cell and animal models correlate with Alzheimer's disease (AD) manifestations. Tryptamine prevented growth of human neuroblastoma. Epithelioids recovered growth in tryptamine-free medium, while neuroblasts died. Tryptamine induced epithelioid differentiation forming synaptic vesicles, neuritic contacts, and TrpRS+ axons in stable sublines. A fraction of epithelioids was adhered to satellite cells via trypsin-resistant interdigitating junctions. Tryptamine stimulated satellite division and differentiation into neurons, transitional cell variants and neuroblasts able to repopulate. Both tryptamine-inhibited and hypoxia-downregulated TrpRS translocates into cytoplasmic extensions. TrpRS is secreted into extracellular space as a free protein or within vesicles extended from cytoplasm and then pinched-off from plasma membrane of tryptamine-treated cells. Extracellular vesicles fuse in congophilic TrpRS+ plaques in tryptamine-treated culture and AD brain. TrpRS prominent immunoreactivity is associated with plasma and vesicle membranes of satellites and AD brain degenerated neurons. Tryptamine-modified mouse brain expresses amyloid and abnormal filaments in extracellular and neuronal plasma membrane vesicles. Radiolabeled tryptamine, tryptophan and serotonin uptake was 10-fold lower in tryptamine-resistant compared to tryptamine-sensitive cells. In both variants, tryptamine uptake exceeded tryptophan uptake within 2-h assuring TrpRS inhibition. Here, tryptophanyl-tRNAtrp deficiency implicates in both neurite growth and termination/collapse. Neurite growth termination prompts TrpRS+ vesicularization. TrpRS+ vesicles contribute in neuronal fragmentation and fibrillar-vesicular congophilic plaques in AD brain.

Differential expression of secretory phospholipases A2 in normal and malignant prostate cell lines: regulation by cytokines, cell signaling pathways, and epigenetic mechanisms.

Upregulation of group IIA phospholipase A(2) (sPLA(2)-IIA) correlates with prostate tumor progression suggesting prooncogenic properties of this protein. Here, we report data on expression of three different sPLA(2) isozymes (groups IIA, V, and X) in normal (PrEC) and malignant (DU-145, PC-3, and LNCaP) human prostate cell lines. All studied cell lines constitutively expressed sPLA(2)-X, whereas sPLA(2)-V transcripts were identified only in malignant cells. In contrast, no expression of sPLA(2)-IIA was found in PrEC and DU-145 cells, but it was constitutively expressed by IFN-gamma in LNCaP and PC-3 cells. Expression of sPLA(2)-IIA is upregulated in PC-3 and in PrEC cell in a signal transducer and activator of transcription-1-dependent manner, but not in LNCaP cell. Additional signaling pathways regulating sPLA(2)-IIA expression include cAMP/protein kinase A, p38 mitogen-activated protein kinase, protein kinase C, Rho-kinase, and mitogen-activated/extracellular response protein kinase / extracellular signal-regulated kinase. No deletions were revealed in the sPLA(2)-IIA gene from DU-145 cells lacking the expression of sPLA(2)-IIA. Reexpression of sPLA(2)-IIA was induced by 5-aza-2'-deoxycytidine demonstrating that DNA methylation is implicated in the regulation of sPLA(2)-II. Together, these data suggest that sPLA(2)-IIA and sPLA(2)-V, but not sPLA(2)-X, are differentially expressed in normal and malignant prostate cells under the control of proinflammatory cytokines; epigenetic mechanisms appear involved in the regulation of sPLA(2)-IIA expression, at least in DU-145 cells.

Mapping and molecular characterization of novel monoclonal antibodies to conformational epitopes on NH2 and COOH termini of mammalian tryptophanyl-tRNA synthetase reveal link of the epitopes to aggregation and Alzheimer's disease.

Tryptophanyl-tRNA synthetase (TrpRS) is an interferon-induced phosphoprotein with autoantigenic and cytokine activities detected in addition to its canonical function in tRNA aminoacylation. The availability of monoclonal antibodies (mAbs) specific for TrpRS is important for development of tools for TrpRS monitoring. A molecular characterization of two mAbs raised in mice, using purified, enzymatically active bovine TrpRS as the inoculating antigen, is presented in this report. These IgG1 antibodies are specific for bovine, human and rabbit but not E. coli TrpRS. Immunoreactivity and specificity of mAbs were verified with purified recombinant hTrpRS expressed in E. coli and TrpRS-derived synthetic peptides. One of the mAbs, 9D7 is able to disaggregate fibrils formed by Ser32-Tyr50 TrpRS-peptide. Epitope mapping revealed that disaggregation ability correlates with binding of 9D7 to this peptide in ELISA and immunocytochemistry. This epitope covers a significant part of N-terminal extension that suggested to be proteolytically deleted in vivo from the full-length TrpRS whereas remaining COOH-fragment possesses a cytokine activity. For epitope mapping of mAb 6C10, the affinity selected phage-displayed peptides were used as a database for prediction of conformational discontinuous epitopes within hTrpRS crystal structure. Using computer algorithm, this epitope is attributed to COOH-terminal residues Asp409-Met425. In immunoblotting, the 6C10 mAb reacts preferably with (i) oligomer than monomer, and (ii) bound than free TrpRS forms. The hTrpRS expression was shown to correlate with growth rates of neuroblastoma and pancreatic cancer cells. Immunohistochemically both mAbs revealed extracellular plaque-like aggregates in hippocampus of Alzheimer's disease brain.

Tryptamine induces tryptophanyl-tRNA synthetase-mediated neurodegeneration with neurofibrillary tangles in human cell and mouse models.

The neuropathological hallmarks of Alzheimer's disease (AD) and other taupathies include neurofibrillary tangles and plaques. Despite the fact that only 2-10% of AD cases are associated with genetic mutations, no nontransgenic or metabolic models have been generated to date. The findings of tryptophanyl-tRNA synthetase (TrpRS) in plaques of the AD brain were reported recently by the authors. Here it is shown that expression of cytoplasmic-TrpRS is inversely correlated with neurofibrillary degeneration, whereas a nonionic detergent-insoluble presumably aggregated TrpRS is simultaneously accumulated in human cells treated by tryptamine, a metabolic tryptophan analog that acts as a competitive inhibitor of TrpRS. TrpRSN- terminal peptide self-assembles in double-helical fibrils in vitro. Herein, tryptamine causes neuropathy characterized by motor and behavioral deficits, hippocampal neuronal loss, neurofibrillary tangles, amyloidosis, and glucose decrease in mice. Tryptamine induced the formation of helical fibrillary tangles in both hippocampal neurons and glia. Taken together with the authors' previous findings of tryptamine-induced nephrotoxicity and filamentous tangle formation in kidney cells, the authors' data indicates a general role of tryptamine in cell degeneration and loss. It is concluded that tryptamine as a component of a normal diet can induce neurodegeneration at the concentrations, which might be consumed along with food. Tryptophan-dependent tRNAtrp aminoacylation catalyzed by TrpRS can be inhibited by its substrate tryptophan at physiological concentrations was demonstrated. These findings indicate that the dietary supplementation with tryptophan as a tryptamine competitor may not counteract the deleterious influence of tryptamine. The pivotal role of TrpRS in protecting against neurodegeneration is suggested, providing an insight into the pathogenesis and a possible treatment of neurodegenerative diseases.