Concise Review: Exciting Cells: Modeling Genetic Epilepsies with Patient‐Derived Induced Pluripotent Stem Cells

Human induced pluripotent stem cell (iPSC) models of epilepsy are becoming a revolutionary platform for mechanistic studies and drug discovery. The skyrocketing pace of epilepsy gene discovery is vastly outstripping the development of in vivo animal models. Currently, antiepileptic drug prescribing to patients with specific genetic epilepsies is based on small‐scale clinical trials and empiricism; however, rapid production of patient‐derived iPSC models will allow for precision therapy. We review iPSC‐based studies that have already afforded novel discoveries in diseases with epileptic phenotypes, as well as challenges to using iPSC‐based neurological disease models. We also discuss iPSC‐derived cardiomyocyte studies of arrhythmia‐inducing ion channelopathies that exemplify novel drug discovery and use of multielectrode array technology that can be translated to epilepsy research. Beyond initial studies of Rett, Timothy, Phelan‐McDermid, and Dravet syndromes, the stage is set for groundbreaking iPSC‐based mechanistic and therapeutic discoveries in genetic epilepsies with the potential to impact patient treatment and quality of life. Stem Cells 2016;34:27–33Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134416/1/stem2203_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134416/2/stem2203.pd

Network structure determines patterns of network reorganization during adult neurogenesis

New cells are generated throughout life and integrate into the hippocampus via the process of adult neurogenesis. Epileptogenic brain injury induces many structural changes in the hippocampus, including the death of interneurons and altered connectivity patterns. The pathological neurogenic niche is associated with aberrant neurogenesis, though the role of the network-level changes in development of epilepsy is not well understood. In this paper, we use computational simulations to investigate the effect of network environment on structural and functional outcomes of neurogenesis. We find that small-world networks with external stimulus are able to be augmented by activity-seeking neurons in a manner that enhances activity at the stimulated sites without altering the network as a whole. However, when inhibition is decreased or connectivity patterns are changed, new cells are both less responsive to stimulus and the new cells are more likely to drive the network into bursting dynamics. Our results suggest that network-level changes caused by epileptogenic injury can create an environment where neurogenic reorganization can induce or intensify epileptic dynamics and abnormal integration of new cells.Comment: 28 pages, 10 figure

Speech Preservation during Language-dominant, Left Temporal Lobe Seizures: Report of a Rare, Potentially Misleading Finding

Purpose: To evaluate the prevalence and mechanism of ictal speech in patients with language-dominant, left temporal lobe seizures. Methods: We retrospectively reviewed the video-EEG telemetry records for the presence of ictal speech in 96 patients with surgically proven left temporal lobe epilepsy and studied the seizure-propagation patterns in three patients who required intracranial EEG recordings for seizure localization. Results: Ictal speech preservation was observed in five patients. One patient's seizures demonstrated rapid propagation of the ictal discharges to the contralateral temporal area where the seizure evolved, resembling a nondominant temporal lobe seizure. The other two patients had ictal discharges that remained confined to the inferomesial temporal areas, sparing language cortex. Conclusions: Preservation of speech in complex partial seizures of language-dominant, left temporal lobe origin is rare. Based on intracranial EEG recordings, the likely mechanism underlying this potentially misleading clinical finding is the preservation of language areas due to limited seizure-propagation patterns.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65559/1/j.1528-1167.2006.00606.x.pd

From network structure to network reorganization: implications for adult neurogenesis

Networks can be dynamical systems that undergo functional and structural reorganization. One example of such a process is adult hippocampal neurogenesis, in which new cells are continuously born and incorporate into the existing network of the dentate gyrus region of the hippocampus. Many of these introduced cells mature and become indistinguishable from established neurons, joining the existing network. Activity in the network environment is known to promote birth, survival and incorporation of new cells. However, after epileptogenic injury, changes to the connectivity structure around the neurogenic niche are known to correlate with aberrant neurogenesis. The possible role of network-level changes in the development of epilepsy is not well understood. In this paper, we use a computational model to investigate how the structural and functional outcomes of network reorganization, driven by addition of new cells during neurogenesis, depend on the original network structure. We find that there is a stable network topology that allows the network to incorporate new neurons in a manner that enhances activity of the persistently active region, but maintains global network properties. In networks having other connectivity structures, new cells can greatly alter the distribution of firing activity and destroy the initial activity patterns. We thus find that new cells are able to provide focused enhancement of network only for small-world networks with sufficient inhibition. Network-level deviations from this topology, such as those caused by epileptogenic injury, can set the network down a path that develops toward pathological dynamics and aberrant structural integration of new cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/85406/1/ph10_4_046008.pd

Prolonged seizures recruit caudal subventricular zone glial progenitors into the injured hippocampus

Neurogenesis persists in the adult rat rostral forebrain subventricular zone (SVZ) and is stimulated by status epilepticus (SE). More caudal SVZ (cSVZ) neural progenitors migrate to the hippocampus after ischemic injury and contribute to CA1 pyramidal cell regeneration. Because SE also damages the hippocampus, we examined the effects of SE on cSVZ precursors. SE was induced in adult rats with pilocarpine, and cell proliferation in cSVZ and hippocampus was examined by bromodeoxyuridine (BrdU) and retroviral reporter labeling. Neural precursors were assayed by immunostaining for specfic markers between 1 and 35 days after SE. BrdU-positive cells labeled prior to SE markedly increased in numbers within 1–2 weeks in the cSVZ and infracallosal region, but not in the corpus callosum. Doublecortin-, polysialic acid neural cell adhesion molecule-, and TUC-4 (TOAD/Ulip/CRMP family-4)-immunostained cells with migrating morphology increased with a similar time course after SE and extended from the cSVZ to CA1 and CA3 regions. Retroviral reporters injected into the cSVZ of controls showed labeled cells with oligodendroglial morphology located in the cSVZ and corpus callosum; when injected 2 days prior to SE, many more reporter-labeled cells appeared several weeks later and were located in the cSVZ, corpus callosum, and hippocampus. Labeled cells showed glial morphologies and expressed astrocyte or oligodendrocyte markers. Neither BrdU- nor retroviral reporter-labeled cells coexpressed neuronal markers in controls or pilocarpine-treated rats. These results indicate that SE increases cSVZ gliogenesis and attracts newly generated glia to regions of hippocampal damage. Further study of seizure-induced gliogenesis may provide insight into mechanisms of adult neural progenitor regulation and epileptogenesis. © 2006 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49285/1/20166_ftp.pd

Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells

Delivery of Proteases in Aqueous Two‐Phase Systems Enables Direct Purification of Stem Cell Colonies from Feeder Cell Co‐Cultures for Differentiation into Functional Cardiomyocytes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/101762/1/1440_ftp.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/101762/2/adhm_201300049_sm_suppl.pd

Scn1b deletion in adult mice results in seizures and SUDEP

Pathogenic lossâ ofâ function variants in SCN1B are linked to Dravet syndrome (DS). Previous work suggested that neuronal pathfinding defects underlie epileptogenesis and SUDEP in the Scn1b null mouse model of DS. We tested this hypothesis by inducing Scn1b deletion in adult mice that had developed normally. Epilepsy and SUDEP, which occur by postnatal day 21 in Scn1b null animals, were observed within 20 days of induced Scn1b deletion in adult mice, suggesting that epileptogenesis in SCN1Bâ DS does not result from defective brain development. Thus, the developmental brain defects observed previously in Scn1b null mice may model other coâ morbidities of DS.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149575/1/acn3785.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/149575/2/acn3785_am.pd

Aberrant seizure-induced neurogenesis in experimental temporal lobe epilepsy

Neurogenesis in the hippocampal dentate gyrus persists throughout life and is increased by seizures. The dentate granule cell (DGC) layer is often abnormal in human and experimental temporal lobe epilepsy, with dispersion of the layer and the appearance of ectopic granule neurons in the hilus. We tested the hypothesis that these abnormalities result from aberrant DGC neurogenesis after seizure-induced injury. Bromodeoxyuridine labeling, in situ hybridization, and immunohistochemistry were used to identify proliferating progenitors and mature DGCs in the adult rat pilocarpine temporal lobe epilepsy model. We also examined dentate gyri from epileptic human hippocampal surgical specimens. Prox-1 immunohistochemistry and pulse-chase bromodeoxyuridine labeling showed that progenitors migrate aberrantly to the hilus and molecular layer after prolonged seizures and differentiate into ectopic DGCs in rat. Neuroblast marker expression indicated the delayed appearance of chainlike progenitor cell formations extending into the hilus and molecular layer, suggesting that seizures alter migratory behavior of DGC precursors. Ectopic putative DGCs also were found in the hilus and molecular layer of epileptic human dentate gyrus. These findings indicate that seizure-induced abnormalities of neuroblast migration lead to abnormal integration of newborn DGCs in the epileptic adult hippocampus, and implicate aberrant neurogenesis in the development or progression of recurrent seizures. Ann Neurol 2005Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/49280/1/20699_ftp.pd

Fibroblast growth factor 2 regulates activity and gene expression of human postâ mitotic excitatory neurons

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature, postâ mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has antiâ depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogeninâ 2 (NEUROG2) to generate a homogenous population of postâ mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline, the vast majority of cells are postâ mitotic, and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f, we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2â responsive genes were determined by RNAâ Seq. FGF2â regulated genes previously identified in nonâ neuronal cell types were upâ regulated (EGR1, ETV4, SPRY4, and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuronâ specific genes were also identified that may mediate FGF2â dependent increases in synaptic efficacy including NRXN3, SYT2, and GALR1. Since several of these genes have been implicated in MDD previously, these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD.Alterations in fibroblast growth factor (FGF) signaling have been implicated in major depressive disorder (MDD). In this article, human stem cells are differentiated into glutamatergic neurons. FGF2 treatment of these neurons increases activity as determined using calcium imaging. RNAseq studies implicate a number of genes in this regulation of neuronal activity by FGF2 including SYT2, NRXN3, and GALR1.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/1/jnc14255-sup-0001-SupInfo.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/2/jnc14255-sup-0002-TableS1-S2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/3/jnc14255.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/143704/4/jnc14255_am.pd