3D-Suspension culture platform for high throughput screening of neurotoxic chemicals using LUHMES dopaminergic neurons.

Three-dimensional (3D) cell culture in vitro promises to improve representation of neuron physiology in vivo. This inspired development of a 3D culture platform for LUHMES (Lund Human Mesencephalic) dopaminergic neurons for high-throughput screening (HTS) of chemicals for neurotoxicity. Three culture platforms, adhesion (2D-monolayer), 3D-suspension, and 3D-shaken, were compared to monitor mRNA expression of seven neuronal marker genes, DCX, DRD2, ENO2, NEUROD4, SYN1, TH, and TUBB3. These seven marker genes reached similar maxima in all three formats, with the two 3D platforms showing similar kinetics, whereas several markers peaked earlier in 2D adhesion compared to both 3D culture platforms. The differentiated LUHMES (dLUHMES) neurons treated with ziram, methylmercury or thiram dynamically increased expression of metallothionein biomarker genes MT1G, MT1E and MT2A at 6 h. These gene expression increases were generally more dynamic in 2D adhesion cultures than in 3D cultures, but were generally comparable between 3D-suspension and 3D-u plate (low binding) platforms. Finally, we adapted 3D-suspension culture of dLUHMES and neural stem cells to 1536 well plates with a HTS cytotoxicity assay. This HTS assay revealed that cytotoxicity IC50 values were not significantly different between adhesion and 3D-suspension platforms for 31 of 34 (91%) neurotoxicants tested, whereas IC50 values were significantly different for at least two toxicants. In summary, the 3D-suspension culture platform for LUHMES dopaminergic neurons supported full differentiation and reproducible assay results, enabling quantitative HTS (qHTS) for cytotoxicity in 1536 well format with a Robust Z' score of 0.68.

LUHMES Dopaminergic Neurons Are Uniquely Susceptible to Ferroptosis.

Ferroptosis is a necrotic cell death caused by lipid oxidation that may be responsible for neural degeneration in Parkinson's disease. We assessed whether three neuronal cell lines are sensitive to killing by ferroptosis. Ferroptosis inducer erastin killed LUHMES neurons at sub-micromolar concentrations, whereas neuronal cells derived from SH-SY5Y cells or neural stem cells were at least 50-fold less sensitive. LUHMES differentiated neurons were likewise sensitive to killing by RSL3 or ML210, inhibitors of the glutathione peroxidase 4 enzyme (GPX4) that consumes GSH to detoxify lipid peroxides. Additional assays showed that erastin, RSL3, and ML210 increased lipid peroxide levels, and that LUHMES neurons were protected from both peroxide accumulation and cell death by ferrostatin-1. A possible role of iron was assessed by evaluating the effects of five metal chelators on cytotoxicity of erastin and RSL3. LUHMES neurons were protected from RSL3 by three of the chelators, 2,3-dimercapto-1-propanesulfonic acid (DMPS), deferoxiprone (DFX), and deferiprone (DFP). Collectively, these results demonstrate the vulnerability of LUHMES neurons to ferroptosis by chemical treatments that disrupt glutathione synthesis, lipid peroxide detoxification, or iron metabolism. The same vulnerabilities may occur in CNS neurons, which reportedly generate low levels of GSH and metallothioneins, limiting their ability to neutralize oxidative stresses and toxic metals. These results suggest a rationale and methods to search for environmental toxicants that may exploit these vulnerabilities and promote neurodegenerative diseases.

P450 Side-Chain Cleavage Enzyme (P450-SCC) Is an Ovarian Autoantigen in a Mouse Model for Autoimmune Oophoritis.

Steroid-producing cells contain key cytochrome P450 enzymes, such as side-chain cleavage (P450-SCC) and 17α-hydroxylase (17α-OH). They are required for steroid hormone synthesis and considered antigens associated with Addison's disease and autoimmune primary ovarian insufficiency (POI). We studied an animal model for human autoimmune POI in mice with autoimmune oophoritis induced by neonatal thymectomy performed at day 3 (TX3). We previously identified an oocyte-specific protein as a major antigen inciting autoimmune oophoritis in mice. In this study, we characterized ovarian steroid-producing cell antigens. Using indirect immunofluorescence staining, we tested immune reactions in mouse ovarian and adrenal tissue sections with sera from TX3 female mice. More than half of the TX3 mice (8 of 15) produced antibodies reacting with both ovarian and adrenal steroid-producing cells, including some that reacted to oocytes as well. We produced recombinant proteins for the three key steroidogenic enzymes 17α-OH, P450-SSC, and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) and tested their immune reactions with individual mouse sera. By immunoblotting, all mouse sera that reacted with the steroid-producing cells (n = 8) were shown to react with the P450-SCC, but not with the 17α-OH or 3ß-HSD recombinant proteins. The sham-operated mouse sera and TX3 mouse sera negative for steroid-producing cells did not react with the P450-SCC recombinant protein. Our findings indicate that the P450-SCC is a specific and unique major antigen within the ovarian steroid-producing cells. Given their similarity of predicted antigenicity, we assume that P450-SCC acts in human autoimmune POI as it does in mouse autoimmune oophoritis.

The MT1G Gene in LUHMES Neurons Is a Sensitive Biomarker of Neurotoxicity.

Identification of toxicants that underlie neurological diseases is a neglected area awaiting a valid strategy to identify such toxicants. We sought biomarkers that respond to known neurotoxicants in LUHMES immortalized neurons and evaluated these biomarkers for use in screening libraries of environmental toxicants. LUHMES immortalized human dopaminergic neurons were surveyed by RNA sequencing following challenge with parkinsonian toxicants rotenone, 6-hydroxydopamine, MPP+, and ziram (zinc dimethyldithiocarbamate; Zn2+DDC2), as well as additional toxicants paraquat, MS275, and methylmercury. The metallothionein gene MT1G was the most dynamic gene expression response to all seven toxicants. Multiple toxicants also increased transcripts for SLC30A1 and SLC30A2 zinc secretion transporters, the SLC7A11 xCT cystine/glutamate antiporter important for glutathione synthesis, DNA damage inducible transcript 3 (DDIT3), and secreted growth factors FIBIN and CXCL12, whereas several toxicants decreased expression of the apelin growth factor (APLN). These biomarker genes revealed stress responses to many toxicants at sub-cytotoxic concentrations. Since several of these biomarker genes and prior neurological disease studies implicated disruption of metal distribution, we tested metal chelator thiram (dimethyldithiocarbamate, DDC), ziram, and several other metals and metal chelates for cytotoxicity and induction of MT1G expression. Metals and chelators that caused dynamic increases in MT1G expression also caused cytotoxicity, except Ni2+DDC2 induced MT1G at 5 µM, but lacked cytotoxicity up to 100 µM. These results bolster prior work suggesting that neurons are characteristically sensitive to depletion of glutathione or to disruption of cellular metal distribution and provide biomarkers to search for such neurotoxicants in chemical libraries.

The Toxmatrix: Chemo-Genomic Profiling Identifies Interactions That Reveal Mechanisms of Toxicity.

A chemical genomics "Toxmatrix" method was developed to elucidate mechanisms of cytotoxicity using neuronal models. Quantitative high-throughput screening (qHTS) was applied to systematically screen each toxicant against a panel of 70 modulators, drugs or chemicals that act on a known target, to identify interactions that either protect or sensitize cells to each toxicant. Thirty-two toxicants were tested at 10 concentrations for cytotoxicity to SH-SY5Y human neuroblastoma cells, with results fitted to the Hill equation to determine an IC50 for each toxicant. Thirty-three toxicant:modulator interactions were identified in SH-SY5Y cells for 14 toxicants, as modulators that shifted toxicant IC50 values lower or higher. The target of each modulator that sensitizes cells or protects cells from a toxicant suggests a mode of toxicant action or cellular adaptation. In secondary screening, we tested modulator-toxicant pairs identified from the SH-SY5Y primary screening for interactions in three differentiated neuronal human cell lines: dSH-SY5Y, conditionally immortalized dopaminergic neurons (LUHMES), and neural stem cells. Twenty toxicant-modulator pairs showed pronounced interactions in one or several differentiated cell models. Additional testing confirmed that several modulators acted through their primary targets. For example, several chelators protected differentiated LUHMES neurons from four toxicants by chelation of divalent cations and buthionine sulphoximine sensitized cells to 6-hydroxydopamine and 4-(methylamino)phenol hemisulfate by blocking glutathione synthesis. Such modulators that interact with multiple neurotoxicants suggest these may be vulnerable toxicity pathways in neurons. Thus, the Toxmatrix method is a systematic high-throughput approach that can identify mechanisms of toxicity and cellular adaptation.

Characterization of three human cell line models for high-throughput neuronal cytotoxicity screening.

More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd.

Ovarian abnormalities in a mouse model of fragile X primary ovarian insufficiency.

FMR1 premutation (PM) alleles have 55-200 CGG·CCG-repeats in their 5' UTR. PM carriers are at risk of fragile X-associated tremor and ataxia syndrome (FXTAS). Females are also at risk for FX primary ovarian insufficiency (FXPOI). PM pathology is generally attributed to deleterious properties of transcripts with long CGG-tracts. For FXPOI, hormone changes suggest a reduced residual follicle pool. Whether this is due to a smaller than normal original follicle pool or an increased rate of follicle depletion is unclear. A FX-PM mouse the authors generated with 130 CGG·CCG-repeats in the endogenous Fmr1 gene recapitulates features of FXTAS. Here the authors demonstrate that the gross development of the ovary and the establishment of the primordial follicle pool is normal in these mice. However, these animals show a faster loss of follicles of all follicle classes, suggesting that the problem is intrinsic to the ovary. In addition, many oocytes show aberrant nuclear accumulation of FMRP and elevated levels of ubiquitination. Furthermore, PM follicles are smaller and have fewer granulosa cells (GCs) than normal. Thus, these animals have ovarian abnormalities involving both the oocytes and GCs that may shed light on the molecular basis of FXPOI in humans.

NLRP5 mediates mitochondrial function in mouse oocytes and embryos.

Unraveling molecular pathways responsible for regulation of early embryonic development is crucial for our understanding of female infertility. Maternal determinants that control the transition from oocyte to embryo are crucial molecules that govern developmental competence of the newly conceived zygote. We describe a series of defects that are triggered by a disruption of maternal lethal effect gene, Nlrp5. Previous studies have shown that Nlrp5 hypomorph embryos fail to develop beyond the two-cell stage. Despite its importance in preimplantation development, the mechanism by which the embryo arrest occurs remains unclear. We confirmed that Nlrp5 mutant and wild-type females possess comparable ovarian germ pool and follicular recruitment rates. However, ovulated oocytes lacking Nlrp5 have abnormal mitochondrial localization and increased activity in order to sustain physiological ATP content. This results in an accumulation of reactive oxygen species and increased cellular stress causing mitochondrial depletion. Compromised cellular state is also accompanied by increased expression of cell death inducer Bax and depletion of cytochrome c. However, neither genetic deletion (Bax/Nlrp5 double knockout) nor mimetic interference (BH4 domain or Bax inhibitory peptide) were sufficient to alleviate embryo demise caused by depletion of Nlrp5. We therefore conclude that lack of Nlrp5 in oocytes triggers premature activation of the mitochondrial pool, causing mitochondrial damage that cannot be rescued by inactivation of Bax.

Autoimmune oophoritis with multiple molecular targets mitigated by transgenic expression of mater.

Primary ovarian insufficiency (POI) resulting from ovarian autoimmunity is a poorly understood clinical condition lacking in effective treatments. Understanding the targets of the autoimmune response and induction of ovarian-specific tolerance would allow development of focused therapies to preserve fertility in an at-risk population. MATER (maternal antigen that embryos require) is a known ovarian autoantigen targeted in autoimmune syndromes of POI. We attempt to induce ovarian-specific tolerance via transgenic expression of the MATER antigen on potentially tolerogenic antigen-presenting cells (APC), which typically present antigen via the major histocompatibility complex (MHC) class II molecule. We hypothesize that expression of MATER in a MHC class II-dependent manner on APC can mediate induction of ovarian tolerance. We utilized a well-characterized murine model of ovarian autoimmunity, whereby oophoritis develops after d 3 neonatal thymectomy (NTx). Wild-type and transgenic mice, carrying an MHC Class II-driven Mater gene (IE-Mater), were subjected to NTx and assessed for evidence of autoimmune oophoritis. After disease induction by NTx, female mice carrying the IE-Mater transgene had significant reductions in histological oophoritis (56%) and circulating ovarian autoantibodies (28%) compared with wild-type females (94% and 82%, respectively). Incidence of other autoimmunity was unaffected as assessed by antinuclear autoantibodies. Transgenic expression of MATER in APC can induce antigen-specific tolerance with a significant reduction in ovarian autoimmunity. Lack of complete disease protection suggests that other antigens may also play a role in autoimmune oophoritis. As a known autoantigen in the human APS1 (autoimmune polyglandular syndrome type 1), which is associated with POI, MATER may represent a relevant target for future diagnostic and therapeutic clinical interventions.

Effect of vitrification and thawing on human oocyte ATP concentration.

Vitrification/thawing has a significant negative impact on oocyte ATP concentration. However, incubating oocytes up to 180 minutes after thawing allowed oocytes to regain some ATP level.

Five mutations of mitochondrial DNA polymerase-gamma (POLG) are not a prevalent etiology for spontaneous 46,XX primary ovarian insufficiency.

The study objective was to determine if mutations in mitochondrial DNA polymerase gamma (POLG) are associated with spontaneous 46,XX primary ovarian insufficiency (sPOI) using restriction fragment length polymorphism analysis of genomic DNA. Of 201 women with 46,XX sPOI analyzed, we found only one case (0.5%, 95% confidence interval 0-3%) of heterozygosity for a POLG mutation, suggesting that this is not a common genetic etiology for this form of infertility.

Investigation of the human stem cell factor KIT ligand gene, KITLG, in women with 46,XX spontaneous premature ovarian failure.

OBJECTIVE: To investigate mutations in the human KIT ligand gene (KITLG) gene as a mechanism of 46,XX spontaneous premature ovarian failure. The human KIT ligand gene, known also as human stem cell factor, is the ligand of the c-kit transmembrane tyrosine kinase receptor (KIT). This ligand-receptor interaction is known to play important roles in mouse germ cell migration and proliferation. DESIGN: Cross-sectional study. SETTING: Clinical research center. PATIENT(S): Forty women with 46,XX spontaneous premature ovarian failure. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Single-stranded conformational polymorphism analysis and DNA sequencing. RESULT(S): We found one nucleotide change of the KITLG coding region (811G-->T) that led to an alteration of the amino acid composition of the KITLG protein in one Caucasian patient (Asp210Tyr). However, we found the same alteration in two normal control Caucasian samples. Three nucleotide substitutions were found in the noncoding exon of KITLG (exon 10). We also identified two intronic polymorphisms. Thus, we did not identify a single significant mutation in the coding region of the KITLG gene in any of 40 patients (upper 95% confidence limit is 7.2%). CONCLUSION(S): Mutations in the coding regions of the KITLG gene appear not to be a common cause of 46,XX spontaneous premature ovarian failure in North American women.

Developmental expression and subcellular localization of mouse MATER, an oocyte-specific protein essential for early development.

We reported previously that Mater is a maternal effect gene that is required for early embryonic development beyond the two-cell stage in mice. Here we show the expressional profile of Mater and its protein during oogenesis and embryogenesis as well as its subcellular localization in oocytes. Mater mRNA was detectable earliest in oocytes of type 2 follicles, whereas MATER protein appeared earliest in oocytes of type 3a primary follicles. Both mRNA and protein accumulated during oocyte growth. In situ hybridization showed that Mater mRNA appeared progressively less abundant in oocytes beyond type 5a primary follicles. By ribonuclease protection assay, Mater mRNA was abundant in germinal vesicle oocytes, but was undetectable in all stages of preimplantation embryos. In contrast, the protein persisted throughout preimplantation development. Immunogold electron microscopic analysis revealed that MATER was located in oocyte mitochondria and nucleoli, and close to nuclear pores. Taken together, our data indicate that Mater gene transcription and protein translation are active during oogenesis, but appear inactive during early embryogenesis. Thus, Mater and its protein are expressed in a manner typical of maternal effect genes. The presence of MATER protein in mitochondria and nucleoli suggests that it may participate in both cytoplasmic and nuclear events during early development.

Ovarian antibodies as detected by indirect immunofluorescence are unreliable in the diagnosis of autoimmune premature ovarian failure: a controlled evaluation.

BACKGROUND: Ovarian antibodies as detected by indirect immunofluorescence have been used to detect ovarian autoimmunity, but to our knowledge the rate of false positive findings using this method has never been reported. METHODS: Here we examine whether a commercially available ovarian antibody test system, using cynomologous monkey ovary, might be useful in the diagnosis of autoimmune premature ovarian failure. The test was performed in a blinded manner in 26 young women with 46,XX spontaneous premature ovarian failure, in 26 control women with regular menstrual cycles (matched for age, race, and parity) and 26 control men (matched for age and race). We also compared the frequency of other autoantibodies associated with ovarian autoimmunity. RESULTS: As a group young women with premature ovarian failure had an increased incidence of thyroid and gastric parietal cell autoimmunity (p < 0.05). Unexpectedly, however, nearly one third (31%) of normal control women had ovarian antibodies using the commercially available test. One half of young women with premature ovarian failure were found to have ovarian antibodies (P = 0.26). In our own laboratory we found similar results and we were unable to improve the specificity of the test. None of 26 men were found to have ovarian antibodies (P < 0.001). CONCLUSION: Since approximately one third of normal women were found to have ovarian antibodies using the system under study, we conclude that ovarian antibodies as detected by this indirect immunofluorescence method have poor specificity. The specificity of any ovarian antibody test should be established before it is used clinically.

Investigation of KIT gene mutations in women with 46,XX spontaneous premature ovarian failure.

BACKGROUND: Spontaneous premature ovarian failure presents most commonly with secondary amenorrhea. Young women with the disorder are infertile and experience the symptoms and sequelae of estrogen deficiency. The mechanisms that give rise to spontaneous premature ovarian failure are largely unknown, but many reports suggest a genetic mechanism in some cases. The small family size associated with infertility makes genetic linkage analysis studies extremely difficult. Another approach that has proven successful has been to examine candidate genes based on known genetic phenotypes in other species. Studies in mice have demonstrated that c-kit, a transmembrane tyrosine kinase receptor, plays a critical role in gametogenesis. Here we test the hypothesis that human KIT mutations might be a cause of spontaneous premature ovarian failure. METHODS AND RESULTS: We examined 42 women with spontaneous premature ovarian failure and found partial X monosomy in two of them. In the remaining 40 women with known 46,XX spontaneous premature ovarian failure we evaluated the entire coding region of the KIT gene. We did this using polymerase chain reaction based single-stranded conformational polymorphism analysis and DNA sequencing. We did not identify a single mutation that would alter the amino acid sequence of the c-KIT protein in any of 40 patients (upper 95% confidence limit is 7.2%). We found one silent mutation at codon 798 and two intronic polymorphisms. CONCLUSION: Mutations in the coding regions of the KIT gene appear not to be a common cause of 46,XX spontaneous premature ovarian failure in North American women.

A human homologue of mouse Mater, a maternal effect gene essential for early embryonic development.

BACKGROUND: Mater is a maternal effect gene required for early embryonic development in mice, and its protein serves as an autoantigen in a mouse model of autoimmune premature ovarian failure. METHODS: Human MATER cDNA was cloned by PCR techniques. The mRNA and protein were determined using hybridization and immunodetection respectively. The cDNA and protein sequences were analysed using bioinformatics software. RESULTS: Human MATER gene spans a approximately 63 kbp DNA at chromosome 19 and is composed of 15 exons and 14 introns. Expression of its mRNA (approximately 4.2 kb) is restricted to the oocytes. Human MATER cDNA (3885 nt) shows an open reading frame (3600 nt) encoding a polypeptide chain composed of 1200 residues with a predicted molecular mass of 134 236 Da. MATER protein (approximately 134 kDa) was detected in human oocytes. The human and mouse cDNA share 67% homology while their deduced polypeptide chains have 53% identity of amino acids. Also, their protein structures have a number of similar features. CONCLUSIONS: The human MATER and mouse Mater genes and proteins are conserved. Characterization of the human MATER and its protein provides a basis for investigating their clinical implications in autoimmune premature ovarian failure and infertility in women.