Investigation of synaptic defects in autism using patient-derived induced pluripotent stem cells

Funding Type: 
Basic Biology III
Grant Number: 
Award Value: 
Disease Focus: 
Neurological Disorders
Stem Cell Use: 
iPS Cell
Public Abstract: 

Autism spectrum disorders (ASD) are a group of neurodevelopmental diseases that occur in as many as 1 in 150 children in the United States. Three hallmarks of autism are dysfunctional communication, impaired social interaction, and restricted and repetitive interests and activities. Even though no single genetic defect has been ascribed to having a causative role in the majority of ASD cases, twin concordance studies and rare familial forms of the disease strongly support a genetic malfunction and a combinatorial effect of genetic risk factors may contribute to the variability in the symptoms. One major obstacle to ASD research is the difficulty in obtaining human neural tissue to model the disease in vitro. Mouse models of ASD are limited since only rare genetic mutations have been identified so far, and single mutations in those genes cannot fully reproduce the range of critical behaviors characteristic of ASD. Direct reprogramming of patient tissues to induced pluripotent stem (iPS) cells and derivation of forebrain neurons from them will provide much needed insight into the molecular mechanism of neuronal dysfunction in diverse individuals on the autism spectrum. The use of patient-derived stem cells to characterize cellular defects brings together two investigative approaches. One is the identification of common cellular and molecular mechanisms that are central to deficiencies across diverse populations of patients. The other is quantitative comparison of pathological features that address differences amongst diverse patients. Our major goal is to characterize the synaptic dysfunction using concrete, quantifiable parameters in human neurons that have specific mutations in key synaptic proteins. This approach will give us a handle into the molecular synaptic complexes that may also be altered in sporadic ASD cases and could help us develop drug strategies that can normalize synaptic function. Although several groups are interested in generating iPS cells from autistic patients, these efforts generally do not have genomic information on the patients, and the large diversity of mutations associated with autism could lead to large variation in synaptic phenotypes. By focusing on generating iPS cells from patients carrying mutations in a small number of critical synaptic proteins and characterizing the molecular components of this complex, we are likely to be in a strong position to identify novel molecular defects associated with autistic synapses. Relative biochemical comparisons of wildtype and mutant protein complexes could help us find ways to restore synaptic function in ASD.

Statement of Benefit to California: 

Many children in California are affected by autism spectrum disorders, which include monogenic syndromes such as Fragile X syndrome and Rett syndrome. However, the majority of cases are idiopathic and an interplay of multiple genetic risk factors is suspected. Since no current drug therapies exist for autism and an accurate diagnosis can only be made in early childhood by largely behavioral criteria, the cost of care and social burden for such a disorder is high, not to mention the devastation to the quality of life for the families of affected children. We would like to identify a core set of proteins found in synapses that are disrupted or dysregulated in autism by a biochemical approach. If we succeed in this effort, we may be able to identify novel biomarkers and molecular targets for specific patient profiles, and by cross-correlating the genetic background to specific behavioral traits in specific individuals, we may come up with molecular targets that are able to address particular symptoms, which should greatly aid in therapeutic regimens that complement existing behavioral therapies. Generating iPS neurons with known copy number variations associated with autism would be a major resource for other laboratories in California and in the field in general. The economic benefit to California is manifold, as many pharmaceutical and biotech companies in California will want to exploit these novel cell lines and the therapeutic targets identified through them in order to design better drugs for autism.

Progress Report: 

The main goal of this project is to establish a cell culture model human neuronal model of autism spectrum disorders (ASD) by generating induced pluripotent stem (iPS) cell lines from patients harboring mutations in genes associated with autism, differentiating them into forebrain neurons, and characterizing their synaptic defects at the cellular and molecular level. We have successfully obtained iPS cells from two autism spectrum disorders, tuberous sclerosis complex (TSC) and Rett syndrome (RTT). We obtained fibroblasts from patients with mutations in the TSC1 and TSC2 genes through the Coriell biorepository. We then reprogrammed them into several TSC patient-specific iPS cell lines. Furthermore, we have obtained male MECP2 mutant iPS cell lines from the lab of Dr. Alysson Muotri to study in parallel with the TSC lines.

We differentiated ASD iPS cell lines into neural progenitor cell (NPCs) and have been examining differences in protein levels and signaling pathways in these cells. Pathway analyses from MECP2 mutant NPCs suggest there may be a marked deficit in several major intracellular signaling pathways, and we are validating those deficits by biochemical analyses and genetic manipulations. Both TSC and RTT forebrain neurons show significant differences in synaptic regulation compared to their respective controls. Alterations in synaptic regulation are being assessed by gene expression analysis, staining for synaptic markers, and electrophysiology. We have made major progress toward realizing our goal of establishing novel iPS cell models for ASD. Furthermore, we obtained very interesting data that should help us elucidate the cell signaling deficits that lead to neuronal dysfunction.

We set out to establish an in vitro human neuronal model of autism spectrum disorders (ASD) by generating induced pluripotent stem (iPS) cell lines from patients harboring specific genetic mutations in syndromic forms of autism, such as Rett Syndrome (RTT) and Tuberous sclerosis (TS). We then differentiated them into neural progenitor cells (NPCs) and forebrain neurons, in order to compare their differentiation potential and to characterize mutation-associated deficits at the cellular and molecular level. Previously published data on cellular and animal models indicate that synaptic deficits are a major feature of the pathophysiology of RTT and TS.

We employed patient-derived induced pluripotent stem cells (iPSCs) from male RTT patients and gender-matched parental controls to probe for functional and molecular deficits in RTT. A similar approach was taken for TS.
As MECP2 is expressed in both the developing and mature central nervous system, we investigated deficits that may arise during early developmental stages (i.e. at the neural progenitor cell or NPC stage), which could then significantly affect neurodevelopmental processes such as neurogenesis and gliogenesis. By quantitative proteomics, we showed that the RTT cells have changes on the molecular level, at both the NPC and neuron stage, compared to their WT control, and that these changes may reflect some of the deficits in the developmental process. We report delays in maturation, such as misregulation of LIN28 at the NPC stage and subsequent deficits in glial differentiation.

Taken together, these results provide a framework for identifying novel early pathways that are perturbed in RTT, as well as potential therapeutics to minimize functional deficits. More generally, it will be of interest to see if these pathways and possible therapeutics may carry over to other related forms of neurodevelopmental disorders, in particular, idiopathic autism.