21 Jun 2017
by Lesley McCollum
In a plenary talk on Tuesday morning, March 28, at the International Congress on Schizophrenia Research meeting in San Diego, Fred Gage of the Salk Institute presented new research demonstrating the usefulness of induced pluripotent stem cells (iPSCs) for modeling schizophrenia. Once a radical idea, iPSCs are now considered a promising new way to investigate cellular development and pathology.
In a complex disorder such as schizophrenia, where genetic code and genomic and cellular mechanisms manifest as high-level impairments, understanding how the disorder originates requires getting a handle on what goes wrong at the most fundamental level. The problem is that many of the suspected mechanisms are out of reach, occurring during cellular development and in brain tissue that is inaccessible. According to Gage, iPSCs have a major advantage here. Cells reprogrammed into iPSCs can be used to generate just about any cell type in the body. This allows researchers to study specific cells relevant to a disease that maintain specific gene variants present in a person with the disease (see SRF related news here, here, and here).
Demonstrating this advantage, Gage presented new research using iPSCs to study how genetic variation contributes to schizophrenia. The study derived iPSCs from pairs of twins where one twin was affected with schizophrenia and the other was not. Cells derived from the affected twins exhibited a decrease in evoked and spontaneous activity compared with controls. Interestingly, the cells from the unaffected twins also exhibited decreased activity, falling into an intermediate level between controls and the affected twins. So the unaffected twin isn’t completely “unaffected,” said Gage. A similar pattern was seen with currents of sodium, fast potassium, and slow potassium channels, where currents were largest in controls and lowest in affected twins.
Using RNA-seq to identify potential genetic mechanisms of the phenotypic differences in the discordant twins, Gage reported alterations in signaling pathways involved in dentate gyrus (DG) development, a region of the hippocampus proposed to be implicated in schizophrenia pathology. According to Gage, this finding highlights the importance of using lineage-specific neurons. RNA-seq also revealed differences in chromatin accessibility, indicating alterations in regulatory regions of genes involved in presynaptic terminals, membrane assembly, and presynaptic membrane organization.
Blood and fibroblasts offer the ability to also identify somatic differences in the twin pairs using DNA-seq. New research investigating shared and unique variance between affected and unaffected twins revealed 33 schizophrenia-specific variants between one set of twins, and six between another. Gage used these variants to create a list of genes, and in future research is looking for variants near candidate genes and pathways that might be leading to defects in physiology. Looking down the road, Gage shared his vision for using CRISPR to mutate specific genes related to channel dynamics to correct the impaired physiology.
Circuits in a dish
In efforts to address an inherent limitation of schizophrenia-in-a-dish models, Gage also shared new progress in building an in vitro hippocampus to put cells into context. His team developed a new protocol to generate human hippocampal CA3 cells in vitro using human parthenogenetic neuronal precursor cells (hpNPCs). The cells were functionally mature and formed synaptic connections with DG cells. As in vivo, hCA3 neurons were distinct from hDG neurons in firing patterns, and the researchers were able to map connections between DG and CA3, demonstrating the beginnings of a hippocampal circuit. They applied this protocol in a new cohort of patients to make CA3 neurons from schizophrenia iPSCs, which differentiated similarly to control cells but showed reduced spontaneous spike rates, suggesting that the spontaneous activity in CA3 and DG-CA3 is impaired in the disorder.
Gage finished with a promising advance that may help make iPSC studies more accessible to other researchers. He presented new work revealing the ability to use Epstein-Barr virus immortalized lymphocytes to generate bipolar disorder iPSCs, which could then be differentiated into DG granule neurons (see SRF news story). The importance of this accomplishment, said Gage, is that many scientists have banks of lymphocytes available and would no longer need to use fibroblasts. Lymphocytes can also be collected with minimal stress to patients, and they maintain patient-by-patient differences.