Bachelor of Arts, University of Illinois at Urbana Champaign (2005)
Doctor of Philosophy, Emory University (2011)
Thomas Sudhof, Postdoctoral Research Mentor
Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here, we used engineered conditional mutations in human neurons and found that heterozygous and homozygous SHANK3 mutations severely and specifically impaired Ih channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih channels reproduced these phenotypes, suggesting that they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) forming Ih channels, indicating that SHANK3 functions to organize HCN channels. Our data suggest SHANK3 mutations predispose to autism, at least partially, by inducing an Ih channelopathy that may be amenable to pharmacological intervention.
View details for DOI 10.1126/science.aaf2669
View details for PubMedID 26966193
Heterozygous mutations of the NRXN1 gene, which encodes the presynaptic cell-adhesion molecule neurexin-1, were repeatedly associated with autism and schizophrenia. However, diverse clinical presentations of NRXN1 mutations in patients raise the question of whether heterozygous NRXN1 mutations alone directly impair synaptic function. To address this question under conditions that precisely control for genetic background, we generated human ESCs with different heterozygous conditional NRXN1 mutations and analyzed two different types of isogenic control and NRXN1 mutant neurons derived from these ESCs. Both heterozygous NRXN1 mutations selectively impaired neurotransmitter release in human neurons without changing neuronal differentiation or synapse formation. Moreover, both NRXN1 mutations increased the levels of CASK, a critical synaptic scaffolding protein that binds to neurexin-1. Our results show that, unexpectedly, heterozygous inactivation of NRXN1 directly impairs synaptic function in human neurons, and they illustrate the value of this conditional deletion approach for studying the functional effects of disease-associated mutations.
View details for DOI 10.1016/j.stem.2015.07.017
View details for Web of Science ID 000364011000010
Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.
View details for DOI 10.1016/j.stemcr.2014.05.020
View details for PubMedID 25254342
The ZC3H14 gene, which encodes a ubiquitously expressed, evolutionarily conserved, nuclear, zinc finger polyadenosine RNA-binding protein, was recently linked to autosomal recessive, nonsyndromic intellectual disability. Although studies have been carried out to examine the function of putative orthologs of ZC3H14 in Saccharomyces cerevisiae, where the protein is termed Nab2, and Drosophila, where the protein has been designated dNab2, little is known about the function of mammalian ZC3H14. Work from both budding yeast and flies implicates Nab2/dNab2 in poly(A) tail length control, while a role in poly(A) RNA export from the nucleus has been reported only for budding yeast. Here we provide the first functional characterization of ZC3H14. Analysis of ZC3H14 function in a neuronal cell line as well as in vivo complementation studies in a Drosophila model identify a role for ZC3H14 in proper control of poly(A) tail length in neuronal cells. Furthermore, we show here that human ZC3H14 can functionally substitute for dNab2 in fly neurons and can rescue defects in development and locomotion that are present in dNab2 null flies. These rescue experiments provide evidence that this zinc finger-containing class of nuclear polyadenosine RNA-binding proteins plays an evolutionarily conserved role in controlling the length of the poly(A) tail in neurons.
View details for DOI 10.1261/rna.043984.113
View details for Web of Science ID 000334677800009
View details for PubMedID 24671764
Available methods for differentiating human embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) into neurons are often cumbersome, slow, and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ESCs and iPSCs can be converted into functional iN cells with nearly 100% yield and purity in less than 2 weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.
View details for DOI 10.1016/j.neuron.2013.05.029
View details for Web of Science ID 000320743400006
Polyadenosine RNA binding proteins (Pabs) play critical roles in regulating the polyadenylation, nuclear export, stability, and translation of cellular RNAs. Although most Pabs are ubiquitously expressed and are thought to play general roles in post-transcriptional regulation, mutations in genes encoding these factors have been linked to tissue-specific diseases including muscular dystrophy and now intellectual disability (ID). Our recent work defined this connection to ID, as we showed that mutations in the gene encoding the ubiquitously expressed Cys3His tandem zinc-finger (ZnF) Pab, ZC3H14 (Zinc finger protein, CCCH-type, number 14) are associated with non-syndromic autosomal recessive intellectual disability (NS-ARID). This study provided a first link between defects in Pab function and a brain disorder, suggesting that ZC3H14 plays a required role in regulating RNAs in nervous system cells. Here we highlight key questions raised by our study of ZC3H14 and its ortholog in the fruit fly Drosophila melanogaster, dNab2, and comment on future approaches that could provide insights into the cellular and molecular roles of this class of zinc finger-containing Pabs. We propose a summary model depicting how ZC3H14-type Pabs might play particularly important roles in neuronal RNA metabolism.
View details for DOI 10.4161/rna.20187
View details for Web of Science ID 000306528800008
View details for PubMedID 22614829
Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.
View details for DOI 10.1073/pnas.1107103108
View details for Web of Science ID 000293129900042
View details for PubMedID 21734151
Methyl-CpG binding proteins (MBDs) are central components of DNA methylation-mediated epigenetic gene regulation. Alterations of epigenetic pathways are known to be associated with several neurodevelopmental disorders, particularly autism. Our previous studies showed that the loss of Mbd1 led to reduced hippocampal neurogenesis and impaired learning in mice. However, whether MBD1 regulates the autism-related cognitive functions remains unknown. Here we show that Mbd1 mutant (Mbd1(-/-)) mice exhibit several core deficits frequently associated with autism, including reduced social interaction, learning deficits, anxiety, defective sensory motor gating, depression and abnormal brain serotonin activity. Furthermore, we find that Mbd1 can directly regulate the expression of Htr2c, one of the serotonin receptors, by binding to its promoter, and the loss of Mbd1 led to elevated expression of Htr2c. Our results, therefore, demonstrate the importance of epigenetic regulation in mammalian brain development and cognitive functions. Understanding how the loss of Mbd1 could lead to autism-like behavioral phenotypes would reveal much-needed information about the molecular pathogenesis of autism.
View details for DOI 10.1093/hmg/ddn102
View details for Web of Science ID 000256978200017
View details for PubMedID 18385101
MicroRNAs (miRNAs) are small non-coding RNAs that inhibit expression of specific target genes at the post-transcriptional level. Sequence variations in miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, could potentially influence the processing and/or target selection of miRNAs. In this study, we have systematically identified single nucleotide polymorphisms (SNPs) associated with 227 known human miRNAs. Among 323 total SNPs that we identify, 12 are located within the miRNA precursor and one is at the eighth nucleotide (+8) of the mature miR-125a, which has been proposed to play a critical role in recognition of mRNA targets by miRNAs. Through a series of in vivo analyses, we unexpectedly find that this miR-125a SNP significantly blocks the processing of pri-miRNA to pre-miRNA, in addition to reducing miRNA-mediated translational suppression. Thus, our study reveals an additional structural requirement for pri-miRNA processing and emphasizes the importance of identifying new miRNA SNPs and their contributions to miRNA biogenesis and human genetic disease.
View details for DOI 10.1093/hmg/ddm062
View details for Web of Science ID 000247475400012
View details for PubMedID 17400653