Primary Cultures Of Human Neurons Research Articles

EMF Antenna Exposure on a Multilayer Human Head Simulation for Alzheimer Disease Treatments.

In this paper, we follow up with our preliminary biological studies that showed that Repeated electromagnetic field stimulation (REMFS) decreased the toxic amyloid-beta (Aβ) levels, which is considered to be the cause of Alzheimer’s disease (AD). The REMFS parameters of these exposures were a frequency of 64 MHz and a Specific absorption rate (SAR) of 0.4 to 0.9 W/Kg in primary human neuronal cultures. In this work, an electromagnetic field (EMF) model was simulated using high-frequency simulation system (HFSS/EMPro) software. Our goal was to achieve the EM parameters (EMF Frequency and SAR) required to decrease the toxic Aβ levels in our biological studies in a simulated human head. The simulations performed here will potentially lead to the successful development of an exposure system to treat Alzheimer’s disease patients. A popular VFH (very high frequency) patch microstrip antenna system was considered in the study. The selection was based on simple and easy construction and appropriateness to the VHF applications. The evaluation of the SAR and temperature distribution on the various head layers, including skin, fat, dura, the cerebrospinal (CSF), and grey matter, brain tissues, were determined for efficacy SAR and safety temperature increase on a simulated human head. Based on a current pulse of 1 A peak current fed to the antenna feeder, a maximum SAR of 0.6 W/Kg was achieved. A range of 0.4 to 0.6 SAR was observed over the various layers of the simulated human head. The initial design of the antenna indicated an antenna size in the order of 1 m in length and width, suggesting a stationary practical model for AD therapy. Future direction is given for wearable antenna and exposure system, featuring high efficiency and patient comfort.

Therapeutic potential of Nlrp1 inflammasome, Caspase-1, or Caspase-6 against Alzheimer disease cognitive impairment.

The sequential activation of Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing protein 1 (Nlrp1) inflammasome, Caspase-1 (Casp1), and Caspase-6 (Casp6) is implicated in primary human neuron cultures and Alzheimer Disease (AD) neurodegeneration. To validate the Nlrp1-Casp1-Casp6 pathway in vivo, the APPSwedish/Indiana J20 AD transgenic mouse model was generated on either a Nlrp1, Casp1 or Casp6 null genetic background and mice were studied at 4-5 months of age. Episodic memory deficits assessed with novel object recognition were normalized by genetic ablation of Nlrp1, Casp1, or Casp6 in J20 mice. Spatial learning deficits, assessed with the Barnes Maze, were normalized in genetically ablated J20, whereas memory recall was normalized in J20/Casp1-/- and J20/Casp6-/-, and improved in J20/Nlrp1-/- mice. Hippocampal CA1 dendritic spine density of the mushroom subtype was reduced in J20, and normalized in genetically ablated J20 mice. Reduced J20 hippocampal dentate gyrus and CA3 synaptophysin levels were normalized in genetically ablated J20. Increased Iba1+-microglia in the hippocampus and cortex of J20 brains were normalized by Casp1 and Casp6 ablation and reduced by Nlrp1 ablation. Increased pro-inflammatory cytokines, TNF-α and CXCL1, in the J20 hippocampus were normalized by Nlrp1 or Casp1 genetic ablation. CXCL1was also normalized by Casp6 genetic ablation. IFN-γ was increased and total amyloid β peptide was decreased in genetically ablated Nlrp1, Casp1 or Casp6 J20 hippocampi. We conclude that Nlrp1, Casp1, or Casp6 are implicated in AD-related cognitive impairment, inflammation, and amyloidogenesis. These results indicate that Nlrp1, Casp1, and Casp6 represent rational therapeutic targets against cognitive impairment and inflammation in AD.

Contemporary circulating enterovirus D68 strains have acquired the capacity for viral entry and replication in human neuronal cells

Background: Enterovirus D68 (EV-D68) is a member of the Picornaviridae family of single-stranded positive-sense RNA viruses. EV-D68 infection has historically been associated with respiratory illnesses in humans. However, in the summers of 2014, 2016, and 2018 EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis (AFM) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. Methods and materials: To assess the neurotropic capacity of EV-D68, we explored the use of the neuroblastoma-derived neuronal cell line, SH-SY5Y, as a cell culture model to determine if differential infection is observed for different EV-D68 isolates. Viral replication and infectivity in SH-SY5Y was assessed using four different assays-infectious virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Results: In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to virus binding and entry were at least partly responsible for the differential infectivity phenotype, since transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains which could not directly infect SH-SY5Y. Conclusion: Taken together, these results suggest that EV-D68 has evolved to acquire novel neurotropic characteristics that may contribute to the development of neurological disease in humans. In addition to supporting virus replication and other functional studies, the cell culture model we’ve developed may help confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.

LncRNA EPIC1 downregulation mediates hydrogen peroxide-induced neuronal cell injury.

Excessive oxidative stress causes neuronal cell injury. Long non-coding RNA (LncRNA) EPIC1 (Lnc-EPIC1) is a MYC-interacting LncRNA. Its expression and potential functions in hydrogen peroxide (H2O2)-stimulated neuronal cells are studied. In SH-SY5Y neuronal cells and primary human neuron cultures, H2O2 downregulated Lnc-EPIC1 and key MYC targets (Cyclin A1, CDC20 and CDC45). Ectopic overexpression of Lnc-EPIC1 increased expression of MYC targets and significantly attenuated H2O2-induced neuronal cell death and apoptosis. Contrarily, Lnc-EPIC1 siRNA potentiated neuronal cell death by H2O2. MYC knockout by CRISPR/Cas9 method also facilitated H2O2-induced SH-SY5Y cell death. Significantly, MYC knockout abolished Lnc-EPIC1-induced actions in H2O2-stimulated neuronal cells. Together, these results suggest that Lnc-EPIC1 downregulation mediates H2O2-induced neuronal cell death.

F-104 Emergence of novel EV-D68 lineages associated with acute flaccid paralysis

Enterovirus D68 (EV-D68) has historically been associated with respiratory illnesses. However, in the summers of 2014 and 2016, EV-D68 outbreaks coincided with a spike in polio-like acute flaccid myelitis/paralysis (AFM/AFP) cases. This raised concerns that EV-D68 could be the causative agent of AFM during these recent outbreaks. To assess the potential neurotropism of EV-D68, we utilized the neuroblastoma-derived neuronal cell line SH-SY5Y as a cell culture model to determine if differential infection is observed for different EV-D68 strains. In contrast to HeLa and A549 cells, which support viral infection of all EV-D68 strains tested, SH-SY5Y cells only supported infection by a subset of contemporary EV-D68 strains, including isolates from the 2014 outbreak. Viral replication and infectivity in SH-SY5Y were assessed using multiple assays: virus production, cytopathic effects, cellular ATP release, and VP1 capsid protein production. Similar differential neurotropism was also observed in differentiated SH-SY5Y cells, primary human neuron cultures, and a mouse paralysis model. Using the SH-SY5Y cell culture model, we determined that barriers to viral binding and entry were at least partly responsible for the differential infectivity phenotype. Transfection of genomic RNA into SH-SY5Y generated virions for all EV-D68 isolates, but only a single round of replication was observed from strains that could not directly infect SH-SY5Y. In addition to supporting virus replication and other functional studies, this cell culture model may help identify the signatures of virulence to confirm epidemiological associations between EV-D68 strains and AFM and allow for the rapid identification and characterization of emerging neurotropic strains.

Induced pluripotent stem cells for disease modeling, cell therapy and drug discovery in genetic autonomic disorders: a review.

The autonomic nervous system (ANS) regulates all organs in the body independent of consciousness, and is thus essential for maintaining homeostasis of the entire organism. Diseases of the ANS can arise due to environmental insults such as injury, toxins/drugs and infections or due to genetic lesions. Human studies and animal models have been instrumental to understanding connectivity and regulation of the ANS and its disorders. However, research into cellular pathologies and molecular mechanisms of ANS disorders has been hampered by the difficulties in accessing human patient-derived ANS cells in large numbers to conduct meaningful research, mainly because patient neurons cannot be easily biopsied and primary human neuronal cultures cannot be expanded.Human-induced pluripotent stem cell (hiPSC) technology can elegantly bridge these issues, allowing unlimited access of patient-derived ANS cell types for cellular, molecular and biochemical analysis, facilitating the discovery of novel therapeutic targets, and eventually leading to drug discovery. Additionally, such cells may provide a source for cell replacement therapy to replenish lost or injured ANS tissue in patients.Here, we first review the anatomy and embryonic development of the ANS, as this knowledge is crucial for understanding disease modeling approaches. We then review the current advances in human stem cell technology for modeling diseases of the ANS, recent strides toward cell replacement therapy and drug discovery initiatives.

Neuroprotective Effect of Myxobacterial Extracts on Quinolinic Acid-Induced Toxicity in Primary Human Neurons.

Quinolinic acid (QUIN) is a neurotoxin, gliotoxin, and proinflammatory molecule involved in the pathogenesis of several neurological diseases. Myxobacteria have been known as a rich source of secondary metabolites with diverse structures and mode of actions. In this study, we examined the potential neuroprotective effects of myxobacterial extracts on QUIN-induced excitotoxicity in primary human neurons. For this purpose, primary cultures of human neurons were pre-incubated with myxobacterial extracts and subsequently treated with QUIN at a pathophysiological concentration of 550nM. The results showed that some myxobacterial extracts can significantly attenuate formation of reactive oxygen species (ROS), nitric oxide (NO) production, and extracellular lactate dehydrogenase (LDH) activity of human neurons. Moreover, myxobacterial extracts were also able to reduce neuronal nitric oxide synthase (nNOS) activity. Some extracts prevented cell death by reducing the activation of poly (ADP-ribose) polymerase (PARP1) by QUIN, therefore by maintaining NAD+ levels. In addition, myxobacterial extracts ameliorated oxidative stress by increasing the intracellular levels of glutathione after treatment with QUIN. The results showed that extracts of Stigmatella sp. UTMC 4072 and Archangium sp. UTMC 4070 and were the most effective in reducing QUIN-induced excitotoxicity in primary human neurons. Due to their antioxidative activity, myxobacterial extracts represent an underexplored source of potential new drugs for the treatment of neurodegenerative diseases.

NEURONAL APOE MODULATES A SPORADIC ALZHEIMER’S DISEASE PHENOTYPE IN PATIENT-DERIVED INDUCED NEURONS

The APOE-ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (sAD) and may be enriched in other clinically relevant populations. While APOE is predominantly expressed by astrocytes in the central nervous system, neuronal expression of APOE is of increasing interest in age-related cognitive impairment, neurological injury, and neurodegeneration. The human and mouse APOE promoter sequences are dissimilar, and contain distinct regions that control expression in a species specific, cell-type specific manner. APOE mRNA and protein is detectable in primary human neuronal cultures in a manner suggestive of de novo protein synthesis. However, neuronal expression of APOE has not been linked to neurotoxicity in a human system. We reprogrammed induced pluripotent stem cells (iPSCs) from three unrelated sAD patients with the ε3/ε4 genotype, used CRISPR/Cas9 gene editing to generate ε3/ε3 isogenic lines, and differentiated them into pure cultures of glutamatergic neurons with no detectable glia. APOE is synthesized and secreted by human induced neurons. Genetic correction of ε4 to ε3 reduces tau phosphorylation and phosphoactivation of a specific kinase. Moreover, ε4 is associated with altered amyloid secretion, and exacerbates the neurotoxic effects of a calcium ionophore. APOE is expressed in human glia-free neuronal culture system, and APOE isoform alters sAD-associated molecular pathways. More detailed mechanistic studies are required to better understand this phenomenon. However, the net effect is that endogenous expression of APOE-ε4 weakens neurons and contributes to the sAD cellular phenotype independently of, but likely cooperatively with, glial APOE.

Differential susceptibility of striatal, hippocampal and cortical neurons to Caspase-6.

Active cysteinyl protease Caspase-6 is associated with early Alzheimer and Huntington diseases. Higher entorhinal cortex and hippocampal Caspase-6 levels correlate with lower cognitive performance in aged humans. Caspase-6 induces axonal degeneration in human primary neuron cultures and causes inflammation and neurodegeneration in mouse hippocampus, and age-dependent memory impairment. To assess whether Caspase-6 causes damage to another neuronal system, a transgenic knock-in mouse overexpressing a self-activated form of Caspase-6 five-fold in the striatum, the area affected in Huntington disease, and 2.5-fold in the hippocampus and cortex, was generated. Detection of Tubulin cleaved by Caspase-6 confirmed Caspase-6 activity. The Caspase-6 expressing mice and control littermates were subjected to behavioral tests to assess Huntington disease-relevant psychiatric, motor, and cognitive deficits. Depression was excluded with the forced swim and sucrose consumption tests. Motor deficits were absent in the nesting, clasping, rotarod, vertical pole, gait, and open field analyzes. However, Caspase-6 mice developed age-dependent episodic and spatial memory deficits identified by novel object recognition, Barnes maze and Morris water maze assays. Neuron numbers were maintained in the striatum, hippocampus, and cortex. Microglia and astrocytes were increased in the hippocampal stratum lacunosum molecular and in the cortex, but not in the striatum. Synaptic mRNA profiling identified two differentially expressed genes in transgenic hippocampus, but none in striatum. Caspase-6 impaired synaptic transmission and induced neurodegeneration in hippocampal CA1 neurons, but not in striatal medium spiny neurons. These data revealed that active Caspase-6 in the striatal medium spiny neurons failed to induce inflammation, neurodegeneration or behavioral abnormalities, whereas active Caspase-6 in the cortex and hippocampus impaired episodic and spatial memories, and induced inflammation, neuronal dysfunction, and neurodegeneration. The results indicate age and neuronal subtype-dependent Caspase-6 toxicity and highlight the importance of targeting the correct neuronal subtype to identify underlying molecular mechanisms of neurodegenerative diseases.

Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine.

Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

Methylene blue inhibits caspases by oxidation of the catalytic cysteine

Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

Measles virus mutants possessing the fusion protein with enhanced fusion activity spread effectively in neuronal cells, but not in other cells, without causing strong cytopathology.

Subacute sclerosing panencephalitis (SSPE) is caused by persistent measles virus (MV) infection in the central nervous system (CNS). Since human neurons, its main target cells, do not express known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), it remains to be understood how MV infects and spreads in them. We have recently reported that fusion-enhancing substitutions in the extracellular domain of the MV fusion (F) protein (T461I and S103I/N462S/N465S), which are found in multiple SSPE virus isolates, promote MV spread in human neuroblastoma cell lines and brains of suckling hamsters. In this study, we show that hyperfusogenic viruses with these substitutions also spread efficiently in human primary neuron cultures without inducing syncytia. These substitutions were found to destabilize the prefusion conformation of the F protein trimer, thereby enhancing fusion activity. However, these hyperfusogenic viruses exhibited stronger cytopathology and produced lower titers at later time points in SLAM- or nectin 4-expressing cells compared to the wild-type MV. Although these viruses spread efficiently in the brains of SLAM knock-in mice, they did not in the spleens. Taken together, the results suggest that enhanced fusion activity is beneficial for MV to spread in neuronal cells where no cytopathology occurs, but detrimental to other types of cells due to strong cytopathology. Acquisition of enhanced fusion activity through substitutions in the extracellular domain of the F protein may be crucial for MV's extensive spread in the CNS and development of SSPE. Subacute sclerosing panencephalitis (SSPE) is a fatal disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). Its cause is not well understood, and no effective therapy is currently available. Recently, we have reported that enhanced fusion activity of MV through the mutations in its fusion protein is a major determinant of efficient virus spread in human neuronal cells and brains of suckling hamsters. In this study, we show that those mutations render the conformation of the fusion protein less stable, thereby making it hyperfusogenic. Our results also show that enhanced fusion activity is beneficial for MV to spread in the CNS but detrimental to other types of cells in peripheral tissues, which are strongly damaged by the virus. Our findings provide important insight into the mechanism for the development of SSPE after MV infection.

Neuroprotective Effects of a Variety of Pomegranate Juice Extracts (PJE) Against the Excitotoxin Quinolinic Acid in Human Primary Neurons.

Quinolinic acid (QUIN) excitotoxicity is mediated by elevated intracellular Ca2+ levels, and nitric oxide (NO•) mediated oxidative stress leading to DNA damage, and cell death due to energy restriction. We evaluated the effect of a series of pomegranate juice extracts (PJE), Helow, Malasi, Qusum, and Hamedh, with antioxidant properties on QUIN induced excitotoxicity on primary cultures of human neurons. We showed that Helow and Malasi can attenuate QUIN-induced excitotoxicity to a greater extent than Qusum and Hamedh from Oman. Similarly, both Helow and Malasi were able to attenuate QUIN-induced Ca2+ influx and nNOS activity to a greater extent compared to Qusum, and Hamedh. All extracts reduced the oxidative effects of increased NO• production, and hence preventing NAD+ depletion and cell death. In addition to the well-known antioxidant properties of these natural phytochemicals, the inhibitory effect of some of these compounds on specific excitotoxic processes such as calcium influx provides additional evidence for the beneficial health effects of PJE in excitable tissue, particularly within the CNS.

Neuroprotective Effects of Rosmarinic Acid on Ciguatoxin in Primary Human Neurons

Ciguatoxin (CTX), is a toxic compound produced by microalgae (dinoflagellate) Gambierdiscus spp., and is bio-accumulated and bio-transformed through the marine food chain causing neurological deficits. To determine the mechanism of CTX-mediated cytotoxicity in human neurons, we measured extracellular lactate dehydrogenase (LDH) activity, intracellular levels of nicotinamide adenine dinucleotide (NAD(+)) and H2AX phosphorylation at serine 139 as a measure for DNA damage in primary cultures of human neurons treated with Pacific (P)-CTX-1B and P-CTX-3C. We found these marine toxins can induce a time and dose-dependent increase in extracellular LDH activity, with a concomitant decline in intracellular NAD(+) levels and increased DNA damage at the concentration range of 5-200nM. We also showed that pre- and post-treatment with rosmarinic acid (RA), the active constituent of the Heliotropium foertherianum (Boraginaceae) can attenuate CTX-mediated neurotoxicity. These results further highlight the potential of RA in the treatment of CTX-induced neurological deficits.

Identification of a Novel Endoplasmic Reticulum Stress Response Element Regulated by XBP1

Understanding the regulatory mechanisms mediating PRNP gene expression is highly relevant to elucidating normal cellular prion protein (PrP) function(s) and the transmissibility of prion protein neurodegenerative diseases. Here, luciferase reporter assays showed that an endoplasmic reticulum stress element (ERSE)-like element, CCAAT-N26-CCACG in the human PRNP promoter, is regulated by ER stress and X-box-binding protein 1 (XBP1) but not by activating transcription factor 6 α (ATF6α). Bioinformatics identified the ERSE-26 motif in 37 other human genes in the absence of canonical ERSE sites except for three genes. Several of these genes are associated with a synaptic function or are involved in oxidative stress. Brefeldin A, tunicamycin, and thapsigargin ER stressors induced gene expression of PRNP and four randomly chosen ERSE-26-containing genes, ERLEC1, GADD45B, SESN2, and SLC38A5, in primary human neuron cultures or in the breast carcinoma MCF-7 cell line, although the level of the response depends on the gene analyzed, the genetic background of the cells, the cell type, and the ER stressor. Overexpression of XBP1 increased, whereas siRNA knockdown of XBP1 considerably reduced, PRNP and ERLEC1 mRNA levels in MCF-7 cells. Taken together, these results identify a novel ER stress regulator, which implicates the ER stress response in previously unrecognized cellular functions.

Human Induced Pluripotent Stem Cell-Derived Models to Investigate Human Cytomegalovirus Infection in Neural Cells

Human cytomegalovirus (HCMV) infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS) cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs), neural progenitor cells (NPCs) and neurons suggests that (i) iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii) Neural stem cells have impaired differentiation when infected by HCMV; (iii) NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv) most iPS-derived neurons are not permissive to HCMV infection; and (v) infected neurons have impaired calcium influx in response to glutamate.

Development of a small anti‐cancer molecule targeting both the intrinsic and extrinsic pathways of apoptosis

Our chemical genetic screen for small molecules that increase lifespan in yeast cellular models of aging by targeting mitochondria‐controlled apoptosis identified lithocholic acid (LCA) as one of such molecules. Aging is one of the major risk factors of cancer, an age‐related disease whose onset can be delayed and incidence reduced by certain pharmacological antiaging interventions. We therefore sought to examine if LCA exhibits an anti‐tumor effect in cultured human cancer cells by activating certain anti‐cancer processes that may play an essential role in cellular aging. We found that LCA, at concentrations that are not cytotoxic to primary cultures of human neurons, kills the neuroblastoma (NB) cell lines BE(2)‐m17, SK‐n‐SH, SK‐n‐ MCIXC and Lan‐1. In the case of BE(2)‐m17, SK‐n‐SH and SK‐n‐ MCIXC cells, the LCA anti‐tumor effect is due to apoptotic cell death. In contrast, the LCA‐triggered death of Lan‐1 cells is not caused by apoptosis. Our findings imply that LCA kills BE(2)‐m17 and SK‐n‐MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase‐9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase‐ 8. Our data suggest a molecular mechanism underlying a potent and selective anti‐tumor effect of LCA in cultured human NB cells. Importantly, we found that a similar mechanism underlies a broad anti‐tumor effect of LCA in cultured mammalian cancer cells derived from different tissues and organisms. Supported by NSERC of Canada.

Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells

Aging is one of the major risk factors of cancer. The onset of cancer can be postponed by pharmacological and dietary anti-aging interventions. We recently found in yeast cellular models of aging that lithocholic acid (LCA) extends longevity. Here we show that, at concentrations that are not cytotoxic to primary cultures of human neurons, LCA kills the neuroblastoma (NB) cell lines BE(2)-m17, SK-n-SH, SK-n-MCIXC and Lan-1. In BE(2)-m17, SK-n-SH and SK-n-MCIXC cells, the LCA anti-tumor effect is due to apoptotic cell death. In contrast, the LCA-triggered death of Lan-1 cells is not caused by apoptosis. While low concentrations of LCA sensitize BE(2)-m17 and SK-n-MCIXC cells to hydrogen peroxide-induced apoptotic cell death controlled by mitochondria, these LCA concentrations make primary cultures of human neurons resistant to such a form of cell death. LCA kills BE(2)-m17 and SK-n-MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase-9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase-8. Based on these data, we propose a mechanism underlying a potent and selective anti-tumor effect of LCA in cultured human NB cells. Moreover, our finding that LCA kills cultured human breast cancer and rat glioma cells implies that it has a broad anti-tumor effect on cancer cells derived from different tissues and organisms.

Lead Dysregulates Serine/Threonine Protein Phosphatases in Human Neurons

It is well established that lead (Pb) exposure in humans leads to learning and memory impairment. However, the biological and molecular mechanisms are still not clearly understood. When over activated, serine/threonine protein phosphatases are known to function as a constraint on learning and memory. Activation of these phosphatases can also result in cytoskeletal changes that will adversely affect learning and memory. We investigated the effects of Pb exposure on these phosphatases in primary cultures of human neurons. Neurons were exposed to physiologically relevant concentrations of Pb (5, 10, 20 and 40 μg/dL) and total phosphatase and PP2A activities were determined in neuronal lysate using para-nitrophenyl phosphate (pNPP), and a PP2A-specific phosphopeptide as substrates. Expression of various serine/threonine phosphatases, tau and its phosphorylation state were determined by Western blot (WB) and immunocytochemistry (ICC). We found that the total phosphatase activity in the neuronal lysate was increased by 30–50% by all the concentrations of Pb tested. PP2A activity was increased by 5 μg/dL Pb only. PP1 expression was increased (ranging from 25–50%) by 10, 20 and 40 μg/dL of Pb. PP2B expression was increased substantially (up to 2.5-fold) by 10 μg/dL Pb, whereas, higher concentrations did not show any effect. On the other hand, Pb (at all concentrations used) decreased expression of PP2A and PP5. Pb exposure induced substantial hyperphosphorylation of tau at serine 199/202 by 5 and 10 μg/dL Pb, and Threonine 231 at higher doses. Expression of total tau was mostly unaffected by lead. Immunocytochemistry data confirmed the WB results of expression of PP1, PP2A, tau protein and the phosphorylation of tau. These results support our hypothesis that Pb exposure up regulates some of the serine/threonine phosphatases (PP1 and PP2B) that are known to impair memory formation, and suggest a novel mechanism of Pb neurotoxicity.