Project Title: The Use of Neuronal Stem Cells in Investigating the Effects of a Cadherin 9/Cadherin 10 mutation in vitro.
Investigator: Wendy Ouriel
Project Summary:
Autism spectrum disorder (ASD) is a class of neurodevelopmental disorders that is primarily diagnosed in young children. Although the cause of AD is unknown, insufficient neural cell adhesion molecules may be a contributing factor11. Cell adhesion is the binding of a cell to another cell or surface, and when cells are linked together, they can communicate with each other via signal transduction. Communication among cells is necessary for the proper growth and development of an organism. In the study, The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells, by Shi et al., the researchers noted that certain genetic mutations in NLGN4X (neuroligin 4) have been associated with autism. NLGN4X is a cell adhesion protein that forms and directs neural synapses; a mutation of this protein is therefore likely to affect neural development. Using neuronal stem cells (NCSs) with a NLGN4X gene knockout, the authors found that NCSs with the knockout showed delayed neuronal development, impaired cell-cell interactions, and downregulation of genes involved in neurodevelopment.
The cadherins are another class of cell adhesion proteins that are also of interest in studying autism disorder. In a genome-wide association study by Wang et al. autistic patients were genotyped to identify genetic risk factors. A significant number of patients had single nucleotide polymorphisms between cadherin 9 and cadherin 10, genes that encode for cell adhesion proteins. However, no further study has been conducted on the role cadherin 9 and cadherin 10 play in the pathogenesis of ASD.
Shi et al. demonstrated that neuronal stem cells are a useful tool to study the affects of knockdown mutation on neurodevelopment in vitro. To study the effects of cadherin 9 and cadherin 10 knockdown on neurodevelopment, NSCs will be used. To investigate changes in neuron morphology, cadherin 9 and cadherin 10 expression will be modulated in NSCs. The NSCs will then be differentiated into mature neurons in vitro, and examined for changes in morphology. To investigate the affect of cadherin 9 and 10 mutation on postsynaptic genes, whole-genome expression analysis will be performed by microarray analysis from the RNA of NSCs upon neuronal differentiation. The overall purpose of performing these two experiments is to use neuronal stem cells to determine how the cadherin 9 and cadherin 10 mutations play a role in the development of ASD.
Relevance:
ASD is a disorder that affects people worldwide3,4. Approximately 1 in 88 children have ASD, with an international prevalence rate of about 1%, according to studies conducted in Asia, Europe, and North America4. The number of people diagnosed with autism has increased over time from about 0.5 per 1,000 people during the 1960’s to 1-2 per 1,000 people in the 2000’s 3,10. The cause of ASD is unknown, but it is hypothesized that genetic mutations leading to neuronal defects may increase ones susceptibility to the disease2,13. Previous studies provided compelling support for the use of stem cells as a tool for studying ASD11,12. This study aims to use neural stem cells to broaden the current understanding of the pathology of ASD.
1. Specific Aim 1
a. Significance
Recent studies have found that patients with autism disorder have a genetic polymorphism of the genes cadherin 9 (CDH9) and cadherin 10 (CDH10) 13. Both of these cadherins are involved in cell-to-cell adhesion of neurons in the brain, which supports many previous hypotheses that autism is caused by neuronal defects. The main component that is missing from this body of research is in vitro evidence that a mutation in these two genes leads to neuronal defects seen in patients with autism disorder. Therefore, the purpose of this study is to induce CDH9 and CDH10 knockdown mutations in NSCs to determine if any morphological similarities exist between the neurons of an autistic patient and the experimental neurons. Shi et al. noted that their knockdown neurons shared similar morphological traits to neurons of autistic patients such as lack of synaptic connections, shorter dendrites, and fewer axons. The researchers also found that fewer neurons formed from the NSCs with the knockdown compared to the control. For this study, a similar morphological analysis will be conducted. NSCs with a CDH9 and CDH10 mutation will be differentiated into neurons in vitro, and monitored over the course of 4 weeks for changes in morphology. The number of neurons that fully mature after four weeks will also be quantified and compared to NSCs without the knockdown mutation. This measure will be taken to determine if a CDH9/10 mutation has any effect on the amount of neurons that survive to a fully differentiated state.
This study is novel because a polymorphism mutation of CDH9 and CDH10 has never been introduced into neural stem cells to study autism disorder. This study will use a mutation in NSCs to look at the way neurons are affected, specifically the morphology of the neurons, such as dendrite length, number of axons, and synaptic connections. This technique can permit researchers to observe, in the lab, the phenotypic affect a cadherin 9/10 mutation has on neurodevelopment. The specific aim of this experiment is to determine if a CDH 9/CDH 10 knockdown affects the neurodevelopment of neural stem cells when differentiated in vitro.
b. Approach
Neural stem cells will be obtained in the manner described in Shi et al. Human dermal fibroblast- derived human induced pluripotent stem cells (hiPSC)(ScienCell) will be obtained. Embryoid bodies will form by separating the hiPSCs colonies, and seeding them on ultra-low binding culture plates (ScienCell) with STEMIUM® human pluripotent stem cell growth medium (ScienCell), and replacing the medium every two days. After 5 days, the medium will be switched to N2 supplement ((DMEM/F12) (Invitrogen)), and 1% penicillin/streptomycin to direct differentiation of the embryoid bodies into neurospheres. After 10 days, the neurospheres can be collected and placed in Matrigel-coated plates with N2 media and 20 ng/mL bFGF. After 5-10 days, neural rosettes should appear on the plates, which are to be broken up and resuspended according to the shi et al. protocol. Neuronal differentiation will be done in N2/B27 (Invitrogen) medium not containing bFGF, and the medium replaced every 2 days for up to 10 days.
To introduce a CDH9 and CDH10 knockdown into the neuronal stem cells, an shRNA vector than can effectively knockdown these genes must be chosen. An shRNA vector targeting CDH9, (Mission shRNA lentiviral transduction particles, Sigma Aldrich) and CDH10 (Mission shRNA lentiviral transduction particles, Sigma Aldrich) will be used. The vector will then be transfected into the neuronal stem cells cultured in a 10cm dish with the transfection agent, polyethylenimine11 (Sigma Aldrich), and medium changed daily.
Use of an shRNA vector also requires a retrovirus to infect the cells, as the RNA strand alone will not be capable of penetrating the nuclear membrane of the cell6. A lentivirus can be used to silence the expression of genes within terminally differentiated cells, such as neurons6 and will therefore be a useful tool for this study. In the manner described in Shi et al. a human TRIPZ lentivirus (Dharmacon) inducible shRNA vector for CDH9 and CDH10 plasmids will be transfected into the cells using polyethylenimine. The sample will be incubated for 2 to 3 days, centrifuged at 28,000 rpm for 2 hours and, and lentiviral particles collected and resuspended in PBS solution and stored at -80o C.
To infect the NSCs, the cells that were held in N2/B27 medium, as described above (about 1.5 mL), will be placed in an Eppendorf tube. The lentivirus that was prepared will be added to this tube, and incubated at 37oC for one hour. The contents of the tube will then be emptied onto a poly-ornithine and fibronectin double-coated 6 cm plate (Sigma Aldrich) containing 3mL of N2/B27 media, and incubated overnight at room temperature. After the incubation period, the cells will be transferred into fresh media, and 1 μg/μl doxycycline will be added to induce expression of the vector11 and changed every two days until the completion of the experiment.
Control neurons will be used to provide a basis of comparison to neurons with the CDH 9/10 mutation. These neurons will be prepared just as the experimental neurons, they will be derived from hiPSC, grown in the same media, and differentiated in the same manner. However, these control cells will not be infected with an shRNA vector, and lentivirus. There will be no knockdown of any gene in these cells. Presumably, these cells will differentiate into neurons in vitro in a manner similar to what would be seen in normal development in vivo.
To observe how the CDH9/CDH10 may affect neuron morphology, the cells will be dyed with a red fluorescent protein (mCherry,Clontech) and observed under a fluroescent microscope at various time points. To do this, the cells will be plated in monolayers according to standard protocol18. Briefly, the cells will be grown on chamber well slides (Qiagen) in N2/B27 media. The cells will be trypsinized to detach adherent cells. The cells will then go through passaging procedure to maintain the culture. After passaging, the number of cells will be quantified using a hemocytometer (Qiagen). Following quantification, the cells can then be dyed, and morphology can be obserbed.
Neurons will be examined and photographed 1,3,7, and 14 days after differentiation. The photographs will be examined for the following characteristics: axon number, dendrite length, and number of connections to other neurons. This data will be compared to the control neurons, which will also be dyed with RFP, and development photographed 1,3,7, 14, and 28 days post-differentiation. The number of neurons that are fully differentiated after four weeks (28 days) will also be quantified, and compared to the control.
2. Specific Aim 2
a. Significance
Previous studies have investigated the effect of a knockdown mutation of particular neuroligin genes on the expression of postsynaptic genes11. Neuroligins are involved in cellular adhesion of presynaptic neurexins at the synaptic junction, and their expression directly impacts the expression of other related genes11. A mutation in CDH9 and CDH10 has been associated with autism spectrum disorder13, yet the exact reasons for this remain unknown. ASD is a complicated illness that is likely to be caused by the mutation of many genes. Therefore, the purpose of this study is to determine if a CDH9 and CDH10 mutation leads to the altered expression of other genes in NSCs when upon differentiation in vitro. In the study by Wang et al., the researchers perfomed a genome-wide association study on people with autism to determine common genetic mutations. This study will expand upon Wang et al. by introducing a specific knockout of a known genetic mutation of autistic patients, a mutation in CDH9 and CDH10, and investigating the effect this knockout on gene expression. To do this, neural stem cells will be obtained from hiPSCs, differentiated into neuronal stem cells in vitro, a CDH9/10 mutation will be introduced via shRNA vector and human lentivirus, and at selected times following neuronal differentiation (1, 3, 7, and 14 days post differentiation, as described previously for measuring morphological changes) whole-genome expression analysis will be performed via microarray from the RNA of the differentiated neurons.
There are over one hundred19 genes that have been associated with ASD. One of the major obstacles in understanding the causes of ASD is determining how these genes may interact with each other. The first step in investigating this matter is to identify the key gene regulators. Peforming microarray analysis will permit us to determine when and where particular genes are turned on or off20. With this information, we can better understand the molecular pathways affected by changed in gene expressio, and how ASD ultimately comes to develop in humans. Understanding the basic mechanisms behind ASD will allow us to develop potential treatments in future studies.
This study is novel because there has not been an experiment performed where the effects of a CDH9/CDH10 mutation were analyzed for its effect on the expression of other genes. It is unlikely that ASD is caused by a mutation in CDH9 and CDH10 alone, despite the high prevalence of this mutation in patients with the disorder. Rather, a mutation in these two genes likely affects the expression of other genes important for neuronal development. This study will help determine what other genes are affected by a CDH9/10 mutation, thus shedding further light on the genes involved in the development of ASD. The specific aim for this study is to investigate the effect of cadherin 9 and 10 mutation on gene expression. Whole-genome expression analysis will be performed by microarray analysis from the RNA of NSCs upon neuronal differentiation.
b. Approach
Neuronal stem cells will be obtained in the same manner as described above, and maintained in the same conditions. The stem cells will also be differentiated into neurons, and a CDH9/CDH10 knockdown introduced in the same manner as described previously. Control neurons will also be obtained in the manner described previously, and differentiated just as the experimental neurons, however no gene knockdown will be performed. Microarray analysis will be performed on the control neurons at the same time intervals as the experimental neurons.
To perform a whole genome expression analysis, the total RNA from the differentiated neurons will be obtained 1,3,7, 14, and 28 days post differentiation. Total RNA from the neurons will be extracted via RNeasy mini kit (Qiagen) in addition to an RNAase-free DNAase kit (Qiagen) to eliminate genomic DNA contamination11. The RNA will be purified and extracted according to standard protocol14.
To perform the microarray analysis, an Illumina TotalPrep RNA Amplification Kit (Ambion Inc.) will be used. cRNA will be synthesized from 250ng of total RNA11. This will be done by reverse transcription with an oligo (dT) primer, and a T7 promoter, which acts as a reverse transcriptase15. Once the cRNA has been synthesized, it will be amplified, and hybridized to a Human12 Expression array11(Illumina) according to standard protocol16. The data obtained will be imported into Illumina GenomeStudio11 to gather the genomic expression information. To reduce the amount of potential error during reading, every cell for its given time point (day 1,14, 24,etc. post differentiation) there will be two additional samples taken for microarray analysis.
The data will be uploaded to the TM4 microarray software suite, which will allow for gene quantification and analysis (17). This software will be used to determine if there is differential expression of particular genes as a result of the CDH9/CDH10 knockdown. The data will be analyzed to determine the genes whose expression has been altered as a result of this knockdown.
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