The development of the nervous system requires the proper differentiation, migration and morphogenesis of neurons. The morphological differentiation of individual neurons and assembly of the trillions of neuronal connections that compose the human nervous system occurs through guided extension of axons and dendrites. Molecular guidance cues in the environment of developing neurons guide neuronal growth cones at the tips of extending axons and dendrites. mTOR-mediated local synthesis of new proteins within growth cones as emerged as an important mechanism that controls axon guidance. Mutations in genes involved local protein synthesis are responsible for several human autism spectrum disorders, including Fragile X syndrome and Tuberous Sclerosis Complex (TSC). While modulation of mTOR-dependent protein synthesis is known to be required downstream of both attractive and repulsive axon guidance in several animal model systems, it is unknown if similar mechanisms function in developing human neurons. This proposal will first test whether human Retinal Ganglion Cells (RGCs) derived from human induced pluripotent stem cells (hiPSCs) use mTOR-mediated protein synthesis to respond to positive and negative axon guidance cues.
In Aim 2 we will test the role of the TSC1/TSC2 complex, which is a key upstream negative regulator of mTOR. For this we will generate new lines of hiPSCs by reprograming fibroblast cells from patients with TSC. While this proposal will focus on RGCs, a wide variety of other cell and neuronal types can be studied using these new hiPSC lines. Therefore, these important new cell lines will be a valuable resource for many investigators and will be made available for distribution through WiCell.
In Aim 3 we will test whether RGCs derived from TSC hiPSCs exhibit abnormal response to axon guidance cues tested in Aim 1.

Public Health Relevance

The development of a functional nervous system requires precise guidance of axons and dendrites to their target locations and establishment of proper synaptic connections. This proposal is focused on understanding how the tuberous sclerosis complex (TSC) proteins function in the regulation of mTOR-dependent protein synthesis in human neurons. By developing methods to compare neurons derived from normal and patient- derived induced pluripotent stem cells, we hope to identify sites for therapeutic intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21NS088477-01
Application #
8749617
Study Section
Neurogenesis and Cell Fate Study Section (NCF)
Program Officer
Mamounas, Laura
Project Start
2014-07-01
Project End
2016-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Neurosciences
Type
Schools of Medicine
DUNS #
City
Madison
State
WI
Country
United States
Zip Code
53715
Short, Caitlin A; Suarez-Zayas, Edwin A; Gomez, Timothy M (2016) Cell adhesion and invasion mechanisms that guide developing axons. Curr Opin Neurobiol 39:77-85
Nichol IV, Robert H; Hagen, Kate M; Lumbard, Derek C et al. (2016) Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions. J Neurosci 36:2267-82
Catlett, Timothy S; Gomez, Timothy M (2016) Division of labor in the growth cone by DSCR1. J Cell Biol 213:407-9
Santiago-Medina, Miguel; Gregus, Kelly A; Nichol, Robert H et al. (2015) Regulation of ECM degradation and axon guidance by growth cone invadosomes. Development 142:486-96
Kerstein, Patrick C; Nichol IV, Robert H; Gomez, Timothy M (2015) Mechanochemical regulation of growth cone motility. Front Cell Neurosci 9:244
Doers, Matthew E; Musser, Michael T; Nichol, Robert et al. (2014) iPSC-derived forebrain neurons from FXS individuals show defects in initial neurite outgrowth. Stem Cells Dev 23:1777-87