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Unraveling the Molecular Mechanism Underlying ALS-Linked Astrocyte

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Unraveling the Molecular Mechanism Underlying ALS-Linked Astrocyte Unraveling the molecular mechanism underlying ALS- linked astrocyte toxicity for motor neurons Burcin Ikiz Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 © 2013 Burcin Ikiz All rights reserved Abstract Unraveling the molecular mechanism underlying ALS-linked astrocyte toxicity for motor neurons Burcin Ikiz Mutations in superoxide dismutase-1 (SOD1) cause a familial form of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder. Transgenic mutant SOD1 rodents capture the hallmarks of this disease, which is characterized by a progressive loss of motor neurons. Studies in chimeric and conditional transgenic mutant SOD1 mice indicate that non-neuronal cells, such as astrocytes, play an important role in motor neuron degeneration. Consistent with this non-cell autonomous scenario are the demonstrations that wild-type primary and embryonic stem cell- derived motor neurons selectively degenerate when cultured in the presence of either mutant SOD1-expressing astrocytes or medium conditioned with such mutant astrocytes. The work in this thesis rests on the use of an unbiased genomic strategy that combines RNA-Seq and “reverse gene engineering” algorithms in an attempt to decipher the molecular underpinnings of motor neuron degeneration caused by mutant astrocytes. To allow such analyses, first, mutant SOD1- induced toxicity on purified embryonic stem cell-derived motor neurons was validated and characterized. This was followed by the validation of signaling pathways identified by bioinformatics in purified embryonic stem cell-derived motor neurons, using both pharmacological and genetic techniques, leading to the discovery that nuclear factor kappa B (NF-κB) is instrumental in the demise of motor neurons exposed to mutant astrocytes in vitro. These findings demonstrate the usefulness of this novel genomic approach to study neurodegeneration and to point to NF-κB as a potential valuable therapeutic target for ALS. Table of Contents Chapter 1: Introduction ................................................................................................................1 1.1. Amyotrophic Lateral Sclerosis ..............................................................................................1 1.2. Familial ALS and SOD1 mutations ......................................................................................5 1.3. Motor neuron death in ALS ................................................................................................23 Chapter 2: Validation of a non-cell autonomous model of ALS using purified embryonic stem cell-derived motor neurons exposed to mutant astrocyte-conditioned medium ......... 42 2.1. Introduction .........................................................................................................................42 2.2. Results .................................................................................................................................50 2.3. Conclusion & Discussion ....................................................................................................64 Chapter 3: Identifying gene alterations germane to the toxicity of mutant astrocytes by elucidating relevant molecular pathways that are dysregulated in motor neurons ..............68 3.1. Introduction .........................................................................................................................68 3.2. Results .................................................................................................................................78 3.3. Conclusion & Discussion ..................................................................................................102 Chapter 4: NF-κB regulates the ALS-linked astrocyte toxicity for motor neurons ............110 4.1. Introduction .......................................................................................................................110 4.2. Results ...............................................................................................................................126 i 4.3. Conclusion & Discussion ..................................................................................................138 Chapter 5: Conclusion and Discussion ....................................................................................143 Chapter 6: Experimental Methods ...........................................................................................155 Chapter 7: References ...............................................................................................................165 Chapter 8: Appendix .................................................................................................................189 8.1. The master regulators inferred by the mouse brain interactome .......................................189 8.2. The Reactome pathways for the 24 vs. 0 hour signature ..................................................225 8.3. The Reactome pathways for the 72 vs. 0 hour signature ..................................................230 8.4. The Reactome pathways for the 168 vs. 0 hour signature ................................................245 8.5. The Reactome pathways for the 72 vs. 24 hour signature ................................................249 8.6. The Reactome pathways for the 168 vs. 72 hour signature ..............................................258 ii List of Figures and Tables Figure 1.1: Schematic representation of upper and lower motor neurons that are affected in ALS Figure 1.2: Neuropathology of ALS Table 1.1: ALS-associated genes Figure 1.3: ALS-causing mutations lie throughout the SOD1 polypeptide Table 1.2: Transgenic mutant SOD1 mouse models Figure 1.4: Time course of clinical and neuropathological events in transgenic mutant SOD1G93A mice Figure 1.5: Illustration of apoptosis in spinal cord of transgenic mutant SOD1G93A mice Figure 1.6: Early increase in stress and UPR pathways in vulnerable motor neurons in transgenic mutant SOD1 mice Figure 1.7: Fas-mediated motor neuron death Figure 1.8: Non-cell autonomous damage caused by mutant SOD1 neighboring non-neuronal cells to motor neurons Figure 1.9: Mutant SOD1 astrocyte-derived toxicity for motor neurons Figure 1.10: Medium conditioned specifically with mutant SOD1 astrocytes kills selectively motor neurons Figure 1.11: Astrocytes derived from sALS and fALS patients cause motor neuron death in vitro Figure 1.12: Focally-transplanted astrocytes are neuroprotective in mutant SOD1 rats Figure 1.13: An alternative IFN-γ/LIGHT pathway-mediated mutant astrocyte-derived motor neuron toxicity mechanism iii Figure 2.1: Differentiation of embryonic stem cell-derived motor neurons Figure 2.2: Brachial and lateral motor column identity of embryonic stem cell-derived motor neurons Figure 2.3: Visualization of primary and embryonic stem cell-derived motor neurons Figure 2.4: Sensitivity of embryonic stem cell-derived motor neurons to mutant astrocyte- released toxicity Figure 2.5: Embryonic stem cell-derived motor neurons die through a Bax-dependent mechanism when co-cultured with mutant astrocyte monolayers Figure 2.6: Embryonic stem cell-derived motor neurons are efficiently purified using Magnetic- Activated Cell Sorting Figure 2.7: Purified embryonic stem cell cultures yield of over 90% motor neurons Figure 2.8: Purified embryonic stem cell-derived motor neurons strongly correlate with other motor neuronal markers Figure 2.9: Exposure to mutant astrocyte-conditioned medium kills purified embryonic stem cell- derived motor neurons in culture over a 7-day period Figure 2.10: Purified embryonic stem cell-derived motor neurons are sensitive to mutant astrocyte-conditioned medium regardless of their cell culture age Figure 2.11: The 50% motor neuron loss upon exposure to mutant astrocyte-conditioned medium is not due to the loss of toxic activity Figure 2.12: 72 hours of mutant astrocyte-conditioned medium exposure is the point of no return for embryonic stem cell-derived motor neurons Figure 3.1: Schematic representation of a RNA-Seq assay iv Table 3.1: Comparison of RNA-Seq versus microarray Figure 3.2: Schematic representation of reverse gene engineering analysis Table 3.2: Replicates per timepoint and treatment for the RNA-Seq assay Figure 3.3: The RNA-Seq experimental design to minimize technical variance Figure 3.4: No strong bias in any of the genes for all of the samples of the RNA-Seq data Figure 3.5: Cluster analyses based on the RNA-Seq data at different treatments and timepoints Table 3.3: Number of differentially expressed genes for each signature Figure 3.6: The mouse total brain interactome is the most biologically relevant for the RNA-Seq data Table 3.4: The top 25 master regulators from the 72 vs. 0 hour signature Table 3.5: The top 10 most differentially expressed genes in the 24 vs. 0 hour signature Table 3.6: The top 10 most enriched pathways in the 24 vs. 0 hour signature Figure 4.1: Components of the NF-κB pathway Figure 4.2: Activation of the NF-κB signaling pathway Figure 4.3: The canonical and non-canonical pathways of NF-κB Figure 4.4: Diverse NF-κB complexes can be involved in opposite effects on neuronal survival Table 4.1: Expression of NF-κB in existing motor neuron gene array data Figure 4.5: The canonical NF-κB pathway is activated in motor neurons exposed to mutant astrocyte-conditioned medium Figure 4.6: Increase in nuclear NF-κB expression in motor neurons
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