SciForum Studying the role of DLGAP1 transcripts in MOL2NET autism - PDF document

MOL2NET , 2016 , 2, 10.3390/mol2net-02-07008 1 http://sciforum.net/conference/mol2net-02 SciForum Studying the role of DLGAP1 transcripts in MOL2NET autism using human neural progenitor stem cells Alexis Tapanes-Castillo 1* , Milagros Mulero 1 , Leana Ramos 1 , Rolain Pierre 1 Jossias Genao 1 , , Carlos Canales 1 , Yoan Rodriguez 1,2 and Derek M. Dykxhoorn 2 1 School of Science, St. Thomas University, Miami Gardens, FL 33054, USA 2 Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL 33136, USA * Author to whom correspondence should be addressed; E-Mail: atapanes-castillo@stu.edu; Tel.: +1-305-474-6905; Fax: +1-305-628-6706. Received: / Accepted: / Published: __________________________________________________________________________________ Abstract: This communication provides a brief review of autism spectrum disorder (ASD), including psychological perspectives and biological insights from whole brain, as well as cellular and genomic studies. Our progress towards conducting a functional study on the role of DLGAP1 RNA transcripts in human glutamatergic neurons derived from ASD and control, non-autistic, induced pluripotent stem cells is also described. ____________________________________________________________________________________________________________________________ Key words: autism (ASD), induced pluripotent stem cell (ipsc), neural progenitor cell (npc), DLGAP1 Introduction Psychology research proposes that behavior Autism Spectrum Disorder (ASD) is a commonly associated with ASD results from neurological condition characterized by two core alterations in several cognitive processes, includ- features: (1) impairments in social interaction ing social cognition and global processing [4]. and communication and (2) the presence of The Social Cognition Deficit theory describes restricted interests and/or repetitive behaviors. aspects of ASD as altered processing of human S pectrum refers to ASD’s numerous clinical stimuli, such as other people’s emotions, in presentations, including variations in contrast to processing of non-human stimuli, intelligence, learning disabilities, compulsive such as objects. The Weak Central Coherence behavior, and speech deficits [1]. Studies in the theory argues the general population is primed USA, Canada, South America, Asia, and Europe for central coherence, integrating details in the identified ASD in 1-2% of their populations [2]. context of a global, meaningful whole. In ASD is 4.5 times more common among males contrast, ASD individuals orient their cognitive than females [3]. processing in a more detail-focused, decontextualized, local manner. Lastly, the

MOL2NET , 2016 , 2, 10.3390/mol2net-02-07008 2 http://sciforum.net/conference/mol2net-02 Complexity Theory suggests individuals with ASD experience cognitive overload when A functional study of DLGAP1 processing tasks of certain complexity. Recently, whole transcriptome sequencing From a biological perspective, several models was performed on cultured cortical neurons, have emerged describing the neuropathology derived from the induced pluripotent stem cells of (ipscs) of individuals with autism and control, non-autistic individuals [6,8]. Three Discs large autism [5]. Studies on brain volume and homolog-associated protein 1 (DLGAP1) gray/ transcripts were differentially expressed. white matter volume have led to a theory that the DLGAP1 is a scaffolding protein, expressed in a brain of autistic individuals undergoes structure termed the post-synaptic density, in accelerated growth during early childhood glutamatergic synapses [9]. A 1.7 fold increase in followed by a deceleration in growth later in life. DLGAP1, a 1.93 fold decrease in DLGAP1 MRI studies on functional connectivity support antisense 1, and a 1.51 decrease in DLGAP1 the premise of global hyperconnectivity in the antisense 2 were observed in ASD neurons brains of ASD individuals. Indeed, altered compared to controls [6]. neurotransmission, caused by an imbalance of The aim of our research is to examine the role excitatory and inhibitory signaling during of DLGAP1 in autism biology. We will study development, is hypothesized to be an underlying how reducing DLGAP1 antisense 1 and 2 cause of autism. transcripts affects ASD and control, non-autistic In the brain, excitatory and inhibitory glutamatergic neurons. While the functions of signaling are primarily mediated by two these transcripts are unknown, one hypothesis is neurotransmitters: glutamate and GABA. that these non-coding RNAs negatively regulate Postnatally, during excitatory signaling, DLGAP1 protein-coding mRNA expression. glutamate transmission results in depolarization Based on the transcriptome data, we hypothesize and excitation of the post-synaptic neuron. that reducing DLGAP1 antisense transcripts in Postnatally, GABA is inhibitory. Its post- control neurons will cause them to develop more synaptic transmission results in like autistic neurons. Conversely, decreasing hyperpolarization, so a stronger signal is required DLGAP1 protein coding mRNA in ASD neurons for future neural excitation. This inhibitory role will ameliorate ASD phenotypes. for GABA is a shift from its prenatal function as Currently, we are culturing ASD and control an excitatory transmitter. Since neurite human neural stem/progenitor cells differentiated outgrowth is dependent on neuronal activity, from ipscs, reprogrammed from skin biopsies. A deficits in prenatal excitatory GABA signaling in protocol, which mimics the steps of neurogenesis an individual with ASD could result in an initial during cortical development, was used to period of reduced neurite outgrowth. A postnatal terminally differentiate the cells into period of excitatory glutamate signaling could glutamatergic neurons [6]. Visible differences then lead to an increased number of “local” were observed in the morphology of ASD circuits, which are hyperactive, due to deficient neurons compared to control neurons. While we postnatal GABA inhibitory signaling. These are still characterizing these differences, our reinforced local circuits would later prevent the findings support a previous report showing elimination of superfluous synapses during significant changes in axon and dendrite length, synaptic pruning [6]. branch count, and process count between ASD On a genetic level, over 700 loci are and control cultures [6]. autism , highlighting ASD’s implicated in To reduce DLGAP1 antisense 1 and 2 levels complexity and heterogeneity [7]. Based on these in ASD and control neurons, we designed six findings, approximately 25% of current autism different short hairpin RNAs (shRNAs). cases have an identifiable genetic cause. shRNAs reduce protein expression levels by Disruptions in several genes involved in synaptic targeting complementary RNA for degradation. development and function, as well as cortical Each shRNA was molecularly cloned into a neuron identity, have been associated with lentiviral vector, and the insertion was verified autism [5,6].

MOL2NET , 2016 , 2, 10.3390/mol2net-02-07008 3 http://sciforum.net/conference/mol2net-02 by DNA sequencing. Lentiviruses carrying the and function, of glutamatergic neurons will be shRNAs and a control shRNA, with no known examined and quantified. This data will provide RNA target, were produced and titered. valuable insight to the role of DLGAP1 antisense Procedures were established to amplify DLGAP1 1 and 2 in glutamatergic neurons during normal antisense 1 and 2 cDNA in preparation for development as well as in cases of ASD. quantifying shRNA-mediated transcript Conflicts of Interest: Authors have no conflicts of reduction utilizing real time reverse transcriptase interest. IRB approvals were attained from the Univ. of PCR. Miami and St. Thomas Univ. Acknowledgments: This work was funded by STEM-TRAC grant PO3C110190. Conclusion: Future experiments will transduce We thank Deliabell Hernandez, Vadym Trokhymchuk, Mang Cing (St. Thomas Univ.); Arielis Ortiz, Claudia the shRNA lentiviruses into ASD and control Martinez-Crespo, Melissa Suarez (Miami Dade College); cortical stem/progenitor cells. The cellular Kinsley Belle and Francelethia Johnson (Univ. of Miami) morphology, differentiation, maturation, and for technical assistance. electrophysiology, as well as synaptic structure 4. Ben-Yosef, D.; Anaki, D., Golan, O. Autism Res. in References press, doi: 10.1022/aur.1676. 1. Hus V.; Pickles A.; Cook E.H. Jr.; Risi S.; Lord C. Biol 5. Ardhanaresswaran, K; Volmar, F. Yale J Biol Med. Psychiatry. 2007 , 61, pp.438-448. 2015 , 88, pp. 5-16. 2. Centers for Disease Control and Prevention. 6. DaRosa, B.A. Open Access Dissertations. 2015 , http:// www.cdc.gov/ncbddd/autism/documents/asdprevalencedat scholarlyrepository.miami.edu/oa_dissertations/1531. 7. Ziats M.N.; Rennert, O.M. Front Genet. 2016 , 7, doi: atable.pdf. 2016 . 10.3389/fgene.2016.00065. 3. Christensen D.L.; et al. MMWR Surveil Summ. 2016 , 65, 8. DaRosa, B.A.; et al. Neurosci Lett. 2012 , 516, pp.9-14. pp.1-23. 9. Sheng, M.; Hoogenraad, CC . Annu Rev Biochem. 2007 , 76, pp.823-847.