Biology subjects

Waxman, E. A.

Publications and source records attributed to Waxman, E. A..

6 recordsLinked to original sources

bioRxiv2025

CRISPR-mediated transcriptional activation as a mutation-independent therapeutic strategy for SYNGAP1-related intellectual disability

Synaptic Ras GTPase-activating protein (SynGAP) regulates synaptic strength and neuronal signaling, with essential roles in cortical development and synaptic plasticity. Heterozygous loss-of-function variants in SYNGAP1 cause SYNGAP1-related intellectual disability (SRID), a severe neurodevelopmental disorder characterized by epilepsy, developmental delay, and autism. SYNGAP1 mutations often result in haploinsufficiency, providing a strong rationale for gene-targeted therapies. However, no treatment currently addresses the underlying genetic cause of SRID. Here, we developed a CRISPR-mediated transcriptional activation (CRISPRa) approach to upregulate the functional Syngap1 allele in a SRID mouse model. CRISPRa activated Syngap1, normalized SynGAP protein expression and downstream signaling, and rescued working memory deficits. We validated the translational potential of this strategy in human induced pluripotent stem cell (hiPSC)-derived excitatory cortical neurons. CRISPRa rescued SYNGAP1 in two distinct loss-of-function variant lines. Together, these findings demonstrate the feasibility of mutation-independent transcriptional activation as a therapeutic approach for SRID and its broader applicability to haploinsufficiency disorders.

neuroscience↗

bioRxiv2023

A novel iPSC model reveals selective vulnerability of neurons in Multiple Sulfatase Deficiency

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD iPSC line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

cell biology↗

bioRxiv2023

Cross-site reproducibility of human cortical organoids reveals consistent cell type composition and architecture

BackgroundReproducibility of human cortical organoid (hCO) phenotypes remains a concern for modeling neurodevelopmental disorders. While guided hCO protocols reproducibly generate cortical cell types in multiple cell lines at one site, variability across sites using a harmonized protocol has not yet been evaluated. We present an hCO cross-site reproducibility study examining multiple phenotypes. MethodsThree independent research groups generated hCOs from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol. scRNA-seq, 3D fluorescent imaging, phase contrast imaging, qPCR, and flow cytometry were used to characterize the 3 month differentiations across sites. ResultsIn all sites, hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions with moderate to high fidelity to the in vivo brain that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and morphology. Differential gene expression showed differences in metabolism and cellular stress across sites. Although iPSC culture conditions were consistent and iPSCs remained undifferentiated, primed stem cell marker expression prior to differentiation correlated with cell type proportions in hCOs. ConclusionsWe identified hCO phenotypes that are reproducible across sites using a harmonized differentiation protocol. Previously described limitations of hCO models were also reproduced including off-target differentiations, necrotic cores, and cellular stress. Improving our understanding of how stem cell states influence early hCO cell types may increase reliability of hCO differentiations. Cross-site reproducibility of hCO cell type proportions and organization lays the foundation for future collaborative prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.

neuroscience↗

bioRxiv2023

Generation of a human Tropomyosin 1 knockout iPSC line

The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line.

cell biology↗

bioRxiv2022

Mapping PTBP splicing in human brain identifies targets for therapeutic splice switching including SYNGAP1

Alternative splicing of neuronal genes is controlled in part by the coordinated action of the polypyrimidine tract binding proteins (PTBP1 and PTBP2). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal, controlling the expression of such targets as DLG4, which encodes PSD95, a protein important in synaptic function whose deficiency causes neurodevelopmental disorders. Here, we fully define the PTBP2 footprint in the human transcriptome using both human brain tissue and neurons derived from human induced pluripotent stem cells (iPSC-neurons). We identify direct PTBP2 binding sites and define PTBP2-dependent alternative splicing events, finding novel targets such as STXBP1 and SYNGAP1, which are synaptic genes also associated with neurodevelopmental disorders. The resultant PTBP2 binding and splicing maps were used to test if PTBP2 binding could be manipulated to increase gene expression in PTBP-targeted genes that cause disease when haploinsufficient. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and non-sense mediated decay. Antisense oligonucleotides that disrupt PTBP binding sites on SYNGAP1 redirect splicing and increase gene and protein expression. Collectively, our data provide a comprehensive view of PTBP2-dependent alternative splicing in human neurons and human cerebral cortex, guiding the development of novel therapeutic tools that may benefit a range of neurodevelopmental disorders.

molecular biology↗

bioRxiv2019

Bach2 is a potent repressor of Nrf2-mediated antioxidant enzyme expression in dopaminergic neurons

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional activator of antioxidant response elements (ARE), which function to increase expression of antioxidant enzymes. Recent works suggests Nrf2 activation is a promising protective mechanism against the progressive neurodegeneration in Parkinsons disease (PD). While Nrf2 inducers show some promising results in animal models of PD, the response is limit in dopaminergic neurons with the protection mostly conferred by surrounding glia. The present study characterizes ARE transcriptional repressors Bach1 and Bach 2 (Broad complex- Tramtrack-Bric-a-brac and Cap n Collar homology, basic leucine zipper transcription factors 1 and 2) as a potential explanation for limited Nrf2 activity in these neurons. The current work identified Bach1 and Bach2 in dopaminergic neurons of the human substantia nigra by immunocytochemical analyses. We further identified Bach2 as a more robust inhibitor of Nrf2 responses. The effects of both Bach1 and Bach2 were dependent on their DNA-binding domains, but the DNA-binding domains did not entirely explain the differences in their relative repressor activities. Using IMR-32 neuroblastoma cells, we found differentiation into a dopaminergic neuronal phenotype resulted in increased Bach2 expression, decreased full-length Bach1 expression, increased truncated Bach1, and blunted Nrf2-mediated responses. Bach inhibitors cobalt protoporphyrin or cadmium chloride, which were more effective against Bach1 than Bach2, did not rescue Nrf2 responses. These results provide a novel mechanism for the lack of antioxidant responses and sensitivity of dopaminergic neurons to oxidative damage, in addition to proposing Bach2 as a novel drug target for the treatment of PD.\n\nSignificanceOxidative stress and damage are unifying contributors to explain the progressive loss of dopaminergic neurons in Parkinsons disease (PD). Although increased transcription of antioxidant enzymes by nuclear factor erythroid 2-related factor 2 (Nrf2) shows potential in combating oxidative damage, Nrf2 protection is mostly conferred by surrounding glia with a relative inability of neurons to mount a protective response. This work characterizes transcriptional repressors Bach1 and Bach2 that inhibit Nrf2 responses and prevent antioxidant enzyme production. This study supports Bach2 as a major factor in preventing antioxidant production in neurons and as a novel drug target to protect against PD.

neuroscience↗