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

A Native Function for RAN Translation and CGG Repeats in Regulating Fragile X Protein Synthesis

Repeat-associated non-AUG-initiated translation of expanded CGG repeats (CGG RAN) from the FMR1 5′-leader produces toxic proteins that contribute to neurodegeneration in fragile X-associated tremor/ataxia syndrome. Here we describe how unexpanded CGG repeats and their translation play conserved roles in regulating fragile X protein (FMRP) synthesis. In neurons, CGG RAN acts as an inhibitory upstream open reading frame to suppress basal FMRP production. Activation of mGluR5 receptors enhances FMRP synthesis. This enhancement requires both the CGG repeat and CGG RAN initiation sites. Using non-cleaving antisense oligonucleotides (ASOs), we selectively blocked CGG RAN. This ASO blockade enhanced endogenous FMRP expression in human neurons. In human and rodent neurons, CGG RAN-blocking ASOs suppressed repeat toxicity and prolonged survival. These findings delineate a native function for CGG repeats and RAN translation in regulating basal and activity-dependent FMRP synthesis, and they demonstrate the therapeutic potential of modulating CGG RAN translation in fragile X-associated disorders

Cortical aPKC kinase activity distinguishes neural stem cells from progenitor cells by ensuring asymmetric segregation of Numb

AbstractDuring asymmetric stem cell division, polarization of the cell cortex targets fate determinants unequally into the sibling daughters, leading to regeneration of a stem cell and production of a progenitor cell with restricted developmental potential. In mitotic neural stem cells (neuroblasts) in fly larval brains, the antagonistic interaction between the polarity proteins Lethal (2) giant larvae (Lgl) and atypical Protein Kinase C (aPKC) ensures self-renewal of a daughter neuroblast and generation of a progenitor cell by regulating asymmetric segregation of fate determinants. In the absence of lgl function, elevated cortical aPKC kinase activity perturbs unequal partitioning of the fate determinants including Numb and induces supernumerary neuroblasts in larval brains. However, whether increased aPKC function triggers formation of excess neuroblasts by inactivating Numb remains controversial. To investigate how increased cortical aPKC function induces formation of excess neuroblasts, we analyzed the fate of cells in neuroblast lineage clones in lgl mutant brains. Surprisingly, our analyses revealed that neuroblasts in lgl mutant brains undergo asymmetric division to produce progenitor cells, which then revert back into neuroblasts. In lgl mutant brains, Numb remained localized in the cortex of mitotic neuroblasts and failed to segregate exclusively into the progenitor cell following completion of asymmetric division. These results led us to propose that elevated aPKC function in the cortex of mitotic neuroblasts reduces the function of Numb in the future progenitor cells. We identified that the acyl-CoA binding domain containing 3 protein (ACBD3) binding region is essential for asymmetric segregation of Numb in mitotic neuroblasts and suppression of the supernumerary neuroblast phenotype induced by increased aPKC function. The ACBD3 binding region of Numb harbors two aPKC phosphorylation sites, serines 48 and 52. Surprisingly, while the phosphorylation status at these two sites directly impinged on asymmetric segregation of Numb in mitotic neuroblasts, both the phosphomimetic and non-phosphorylatable forms of Numb suppressed formation of excess neuroblasts triggered by increased cortical aPKC function. Thus, we propose that precise regulation of cortical aPKC kinase activity distinguishes the sibling cell identity in part by ensuring asymmetric partitioning of Numb into the future progenitor cell where Numb maintains restricted potential independently of regulation by aPKC

Changes in Notch signaling coordinates maintenance and differentiation of the Drosophila larval optic lobe neuroepithelia

A dynamic balance between stem cell maintenance and differentiation paces generation of post‐mitotic progeny during normal development and maintenance of homeostasis. Recent studies show that Notch plays a key role in regulating the identity of neuroepithelial stem cells, which generate terminally differentiated neurons that populate the adult optic lobe via the intermediate progenitor cell type called neuroblast. Thus, understanding how Notch controls neuroepithelial cell maintenance and neuroblast formation will provide critical insight into the intricate regulation of stem cell function during tissue morphogenesis. Here, we showed that a low level of Notch signaling functions to maintain the neuroepithelial cell identity by suppressing the expression of pointedP1 gene through the transcriptional repressor Anterior open. Increased Notch signaling, which coincides with transient cell cycle arrest but precedes the expression of PointedP1 in cells near the medial edge of neuroepithelia, defines transitioning neuroepithelial cells that are in the process of acquiring the neuroblast identity. Transient up‐regulation of Notch signaling in transitioning neuroepithelial cells decreases their sensitivity to PointedP1 and prevents them from becoming converted into neuroblasts prematurely. Down‐regulation of Notch signaling combined with a high level of PointedP1 trigger a synchronous conversion from transitioning neuroepithelial cells to immature neuroblasts at the medial edge of neuroepithelia. Thus, changes in Notch signaling orchestrate a dynamic balance between maintenance and conversion of neuroepithelial cells during optic lobe neurogenesis. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94272/1/20995_ftp.pd

Acid-sensing ion channel-1a in the amygdala, a novel therapeutic target in depression-related behavior

No animal models replicate the complexity of human depression. However, a number of behavioral tests in rodents are sensitive to antidepressants and may thus tap important underlying biological factors. Such models may also offer the best opportunity to discover novel treatments. Here, we used several of these models to test the hypothesis that the acid-sensing ion channel-1a (ASIC1a) might be targeted to reduce depression. Genetically disrupting ASIC1a in mice produced antidepressant-like effects in the forced swim test, the tail suspension test, and following unpredictable mild stress. Pharmacologically inhibiting ASIC1a also had antidepressant-like effects in the forced swim test. The effects of ASIC1a disruption in the forced swim test were independent of and additive to those of several commonly used antidepressants. Furthermore, ASIC1a disruption interfered with an important biochemical marker of depression, the ability of stress to reduce BDNF in the hippocampus. Restoring ASIC1a to the amygdala of ASIC1a-/- mice with a viral vector reversed the forced swim test effects, suggesting that the amygdala is a key site of ASIC1a action in depression-related behavior. These data are consistent with clinical studies emphasizing the importance of the amygdala in mood regulation, and suggest that ASIC1a antagonists may effectively combat depression. Copyright © 2009 Society for Neuroscience