1.Identification of mislocalized presynaptic mRNAs in Fragile X

Fragile X Syndrome, the most prevalent genetic form of autism, is caused by a single gene mutation leading to the loss of Fragile X Mental Retardation Protein (FMRP). Normally FMRP binds and transports numerous mRNA along dendrites and axons to their connections (synapses) where they are then made available for translation into proteins. In Fragile X the loss of FMRP alters mRNA translation at synapses, resulting in synaptic dysfunction, impaired cognitive function, and autistic-like behavior. Studies of synaptic translation have relied on rodent model systems. Little is known about local translation at human synapses. In this project we will, for the first time, identify the mRNA population targeted to the presynaptic compartment of human neurons. We are using neurons differentiated from human induced pluripotent stem cells (iPSCs)—human skin cells reprogrammed into stem cells. We rely on microfluidic technology developed by the lead investigator to isolate presynaptic terminals from neurons-derived from human iPSCs from both normal and Fragile X individuals (Taylor et al. J Neurosci 2009). We are focusing on the presynaptic compartment because new findings from our lab and others suggest that translation within this compartment may play a larger role in regulating synaptic transmission than once thought (Taylor et al. J Neurosci 2013). By identifying and comparing the presynaptic mRNA population in normal and Fragile X individuals we expect to identify candidate transcripts that may regulate the cognitive and behavioral changes exhibited with this disorder. In addition to identifying potential targets for therapeutic intervention in autism, this work will have a broad impact in understanding proper synapse development and function in the brain.

Funding: Simons Foundation SFARI Explorer

2.Effect of electrical stimulation on central axon regeneration, synapse re-formation, and plasticity

Deep brain stimulation is used to treat essential tremor, Parkinson’s disease, chronic pain, depression, and multiple other diseases and disorders. The mechanisms by which this activity enhances function are not clearly known. In addition, how activity affects axonal regeneration and synapse re-formation in the CNS is not known. In this project we are using novel methods to target electrodes to axons and synapses to study how activity affects their growth and development and how these properties are altered following injury. We are using gallium and gallium alloys which are liquid at room temperature to flow electrodes into microfluidic channels simplifying the fabrication and alignment of electrodes (Hallfors et al. Lab Chip 2013). These devices may also lead to the future development of flexible neural probes for neuro-stimulation .

Funding: NICHD

3.Development of assays to screen compounds that promote synapse formation/re-formation

High-throughput assays are desperately needed to screen for drugs that promote axon regeneration and synapse re-formation in the CNS. There are numerous challenges associated with the development of these assays, including low yield of primary (post-mitotic) neurons and long culture times needed for neuron maturation. We are using microfluidic approaches to develop these assays (Taylor et al. Nat Methods 2005; Taylor et al. Neuron 2010; Taylor and Jeon Current Opinion in Neurobiology 2010; Taylor and Jeon Crit Rev Biomed Eng 2011). We are using a variety of read-outs to measure the extent of synapse re-formation, including live cell dyes to measure calcium dynamics and synaptic vesicle recycling (indicators of synaptic function). We are exploiting the scalability of microfluidic approaches to generate medium- to high- throughput approaches to test libraries of compounds.

Funding: NICHD

Hallfors, N, A Khan, MD Dickey and AM Taylor (2013). Integration of pre-aligned liquid metal electrodes for neuronal stimulation within an easy-to-assemble microfluidic platform Lab Chip.

Taylor, AM, NC Berchtold, VM Perreau, CH Tu, N Li Jeon and CW Cotman (2009). Axonal mRNA in uninjured and regenerating cortical mammalian axons J Neurosci 29(15): 4697-4707.

Taylor, AM, J Wu, HC Tai, and EM Schuman (2013). Axonal translation of beta-catenin regulates synaptic vesicle dynamics J Neurosci 33(13): 5584-9.

Taylor, AM, M Blurton-Jones, SW Rhee, DH Cribbs, CW Cotman and NL Jeon (2005). A microfluidic culture platform for cns axonal injury, regeneration and transport Nat Methods 2(8): 599-605.

Taylor, AM, DC Dieterich, HT Ito, SA Kim and EM Schuman (2010). Microfluidic local perfusion chambers for the visualization and manipulation of synapses Neuron 66(1): 57-68.

Taylor, AM and NL Jeon (2010). Micro-scale and microfluidic devices for neurobiology Current Opinion in Neurobiology 20(5): 640-647.

Taylor, AM and NL Jeon (2011). Microfluidic and compartmentalized platforms for neurobiological research Crit Rev Biomed Eng 39(3): 185-200.