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  • Addiction, Drugs
  • Information from Lay-Language Summaries is Embargoed Until the Conclusion of the Scientific Presentation

    028—Synapse Formation: CNS I

    Saturday, November 09, 2013, 1:00 pm - 5:00 pm

    28.19: Modeling synaptic pathophysiology in tuberous sclerosis using human pluripotent stem cell-derived neurons

    Location: Halls B-H

    S. AIGNER1, V. COSTA1, B. ATAMAN2, G. BOULTING2, M. VUKCEVIC3, S. ZOFFMANN4, E. SAUTER1, F. KNOFLACH1, M. T. MILLER1, F. BOUKHTOUCHE1, C. GOELDNER1, M. EBELING5, C. PATSCH4, C. A. MEYER4, M. GRAF4, J. BISCHOFBERGER3, J. MOSBACHER1, S. JESSBERGER6, M. E. GREENBERG2, A. GHOSH1, *R. K. JAGASIA1;
    1F. Hoffmann - La Roche Ltd. Pharma Res. and Early Development, Neurosci., Basel, Switzerland; 2Dept. of Neurobiology, Harvard Med. Sch., Boston, MA; 3Dept. of Biomedicine, Univ. of Basel, Basel, Switzerland; 4F. Hoffmann - La Roche Ltd., Pharma Res. and Early Development, Discovery Technologies, Basel, Switzerland; 5F. Hoffmann - La Roche Ltd., Pharma Res. and Early Development, Translational Technol. and Bioinformatics, Basel, Switzerland; 6Brain Res. Institute, Univ. of Zurich, Zurich, Switzerland

    Abstract Body: Several monogenic forms of autism spectrum disorder (ASD) are caused by mutations in genes functioning in the mTOR pathway, leading to translational dysregulation and altered synaptic signaling. TSC1 and TSC2 code for the mTOR negative regulators hamartin and tuberin, respectively, and mutation of either gene causes tuberous sclerosis, a multi-system disorder with high prevalence of ASD. Using zinc finger nuclease-mediated genome editing, we have generated human embryonic stem cell (hESC) lines with heterozygous and homozygous disruption of TSC2. Upon neuralization, neural tube-like rosettes from TSC2-/- hESCs show changes in higher-order structural organization, suggesting early cellular specification dysfunction. In order to identify synaptic signaling defects on the network level, we have developed a protocol to differentiate rosette-derived neuroepithelial precursor cells towards neuronal cultures with functional inhibitory and excitatory synapses. These neurons exhibit several synaptic properties relevant to ASD pathophysiology, including synchronous network activity, synaptic scaling and mGluR5-dependent long-term depression (LTD). We are currently investigating alterations in synaptic signaling properties of TSC2+/- and TSC2-/- neurons. We envision that our human cellular models of neuronal networks will be instrumental in elucidating the mechanisms underlying synaptic dysfunction in ASD and other neurodevelopmental disorders.

    Lay Language Summary: Tuberous sclerosis is a multi-organ disorder that affects about 1 in 6,000 people. It is caused by mutations in either of the tuberous sclerosis complex genes TSC1 or TSC2. In order to better understand the central nervous system manifestations of this debilitating condition, we genetically engineered human embryonic stem cells to carry a mutation in TSC2. When we pushed these cells to become functioning neuronal cells, we found that these tuberous sclerosis neurons had several defects that are relevant to the disease.
    When given the proper cues, pluripotent stem cells develop into different types of cells in the petri dish, including neurons. By means of special enzymes called zinc finger nucleases, researchers can also precisely edit stem cell genomes and introduce specific mutations. We took advantage of this recently developed tool to mutate the TSC2 gene and devised a procedure to turn stem cells into neurons that show many properties of those in the human brain. For example, we saw that our neurons were synaptically connected and spontaneously fired in a synchronous fashion.
    TSC1 and TSC2 proteins normally keep cell proliferation in check and, by controlling protein production and recycling, help maintain proper levels of proteins within the cells. When TSC1 or TSC2 protein is inactivated or reduced due to a lesion in either gene, cell division and protein accumulation can get out of control. Tuberous sclerosis patients therefore develop benign tumors, called hamartomas, in several organs including the brain. Indeed, on their way to developing into neurons, our model cells divided more rapidly and had larger cell bodies in the petri dish. Thus, they recapitulate an important clinical brain manifestation of the disorder.
    Tuberous sclerosis patients very frequently suffer from epileptic seizures and often show autistic behaviors. Researchers believe that this is because excessive amounts of protein at neuronal synapses may alter their structures and perturb communication between neurons. We found that our developing tuberous sclerosis neurons were able to compensate for the reduction of TSC2 protein, whereas maturing neurons that begun to form functional synapses became increasingly sensitive to the precise amount of the protein. We are now addressing to what extent mature tuberous sclerosis neurons show defects in their capacity to form and maintain synaptic connections with each other.
    Our results suggest that by using engineered human stem cell-derived neurons and by closely recapitulating human brain development in the petri dish, we can mirror several cardinal features of tuberous sclerosis. We strongly believe that this will deepen our understanding of the precise molecular mechanisms behind the defects of this genetic disorder. Since there are currently no drugs to adequately manage it, we also believe that our model cells will be critical in the development of therapeutic strategies to cure this serious disorder.
    In addition, it is becoming increasingly clear that problems with regulation of protein levels at neuronal synapses may be critically involved in the development of autism. Although the genetic underpinnings of most cases of autism are unclear, our work may provide insights into the understanding and treatment of this neurodevelopmental disorder as well.