Neurons generated by direct conversion of fibroblasts reproduce synaptic phenotype caused by autism-associated neuroligin-3 mutation

Abstract

Recent studies suggest that induced neuronal (iN) cells that are directly transdifferentiated from nonneuronal cells provide a powerful opportunity to examine neuropsychiatric diseases. However, the validity of using this approach to examine disease-specific changes has not been demonstrated. Here, we analyze the phenotypes of iN cells that were derived from murine embryonic fibroblasts cultured from littermate wild-type and mutant mice carrying the autism-associated R704C substitution in neuroligin-3. We show that neuroligin-3 R704C-mutant iN cells exhibit a large and selective decrease in AMPA-type glutamate receptor-mediated synaptic transmission without changes in NMDA-type glutamate receptor-or in GABA A receptor-mediated synaptic transmission. Thus, the synaptic phenotype observed in R704C-mutant iN cells replicates the previously observed phenotype of R704C-mutant neurons. Our data show that the effect of the R704C mutation is applicable even to neurons transdifferentiated from fibroblasts and constitute a proof-of-concept demonstration that iN cells can be used for cellular disease modeling. induced pluripotent stem (iPS) cells now can be generated from nearly every somatic cell type (1-3). Moreover, efficient protocols now are available to guide the differentiation of iPS cells and embryonic stem (ES) cells into different types of neurons that then can be used for disease modeling (4-7). As a further development, we and others recently described direct conversion of nonneural cells into induced neuronal (iN) cells, which accelerates the generation of neurons from nonneuronal cells and renders this process more uniform and less variable (8-16). The iN cells behave like functional neurons in that they display neuron-like morphologies, express neuronal marker genes, fire action potentials (APs), and, at least in some instances, are not only the postsynaptic recipients of input synapses but also form presynaptic output synapses onto each other or onto cocultured primary neurons.The in vitro generation of neurons from nonneuronal cells is of great interest because of its potential for studying neural development and for producing neurons for regenerative medicine. A possibly even more important application may be the use of neurons transdifferentiated from nonneuronal cells for examining disease mechanisms, because transdifferentiated neurons can be obtained from living patients. Indeed, a large number of studies reported a multitude of disease-related changes in neurons differentiated from patient-derived iPS cells (17)(18)(19)(20), and even iN cells have been used for this purpose (13). However, to date no study has tested whether the neurons directly reprogrammed from fibroblasts, ES cells, or other nonneural cells actually can recapitulate a phenotype observed in primary neurons from the same organism.To address this important question, we asked whether iN cells derived from a mutant mouse with a defined synaptic phenotype can recapitulate the effect. We focused on a previously characterized autism-assoc...

Synaptic Neurexin Complexes: A Molecular Code for the Logic of Neural Circuits

“…Does neurexin- or MDGA-binding activate neuroligins, and if so, does this activation trigger a signal-transduction cascade that involves intracellular neuroligin interactions (e.g., with PSD-95; Irie et al, 1997), or an extracellular cis-interaction? At present, the only evidence for a role of neuroligin cytoplasmic sequences in a physiological function that does not involve overexpression is the gain-of-function effect of the R704C Nlgn3 mutation that produces a large decrease in AMPAR- but not NMDAR-mediated synaptic responses (Etherton et al, 2011b; Chanda et al, 2013). Similarly, it is unclear by what mechanism Nlgn1 is required separately for NMDAR-level maintenance and for postsynaptic LTP, a continuation of the puzzle posed by the requirement for Nrxn3 in LTP.…”

The balancing act of GABAergic synapse organizers

“…Moreover, postnatal transplantation of MGE-derived GABAergic precursor cells reduces seizure activity in the hippocampus [109], suggesting that inhibitory interneuron grafting-based cell therapy could be a powerful therapeutic approach for some brain disorders. In addition to the available animal models, patient-derived induced neuronal (iN) cells can be used to model specific neural circuit dysfunctions that offer new insight into the pathological mechanisms of human brain disorders [110], especially because iN cells derived from embryonic fibroblasts of NL-3 R704C KI mice successfully recapitulated cellular and electrophysiological phenotypes of these mice [111]. However, caution should be used when seeking to match in vivo brain developmental processes using human neural progenitor cells, especially for potential clinical applications [112].…”

The Human Model: Changing Focus on Autism Research

“…Interestingly, the increase in tyrosine hydroxylase was rescued after treatment with roscovitine, an L-type channel blocker, emphasizing the potential for a drug-screening platform using TS-derived neurons. Neuroligins, a class of genes previously implicated in ASD, were also targeted for disease modeling using reprogramming techniques(53, 82). …”