Biology subjects

Pruett-Miller, S. M.

Publications and source records attributed to Pruett-Miller, S. M..

At least 19 recordsLinked to original sources

bioRxiv2026

Myogenic and neural transcriptional circuitry determines PAX3::FOXO1 driven initiation of rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) is largely characterized by the PAX3::FOXO1 (P3F) fusion oncoprotein, yet the mechanisms by which P3F initiates tumorigenesis and cell identity remain incompletely understood. Here, we describe a doxycycline inducible, degradable PAX3::FOXO1 iPSC derived ARMS model (ddP3F) to interrogate the temporal requirements of P3F for ARMS cell identity and proliferation. We show that, while P3F is essential for initiating ARMS transformation from precursor cells, continued expression of P3F is not required for maintaining proliferation, viability, or RMS associated transcriptional programs. Integration of CUT&RUN, bulk RNA seq, and 10X multiome profiling during endothelial-directed differentiation revealed that P3F rapidly pioneers a novel enhancer landscape within two days of expression, activating transcriptional circuits anchored by both specific myogenic and neural transcription factors (TFs), including MYOD1, PRDM12, and POU4F1 (BRN3A). Our single-cell analysis shows that these TFs are specifically co-expressed with P3F from the earliest stages of transformation. Knockout of MYOD1 or POU4F1 impairs ARMS identity acquisition without preventing transformation, whereas PRDM12 loss enhances P3F expression programs and myogenic differentiation. Together, these findings underscore a previously unappreciated interplay between myogenic and neural transcriptional programs during ARMS initiation. Furthermore, these data demonstrate that P3F establishes a stable oncogenic state that persists even after removal, thus providing a framework for evaluating P3F dependent and P3F independent therapeutic vulnerabilities.

cancer biology↗

bioRxiv2026

Biotin-operated ligand trapping enables dynamic spatial redistribution of protein and organelle in living cells

Controlling protein localization through chemical biology tools is essential for mechanistic interrogations, functional perturbations, and therapeutic development. While existing tools cover a wide range of applications, no single approach simultaneously enables inducible trapping, controlled release, and visualization of a protein of interest (POI). To address this gap, we developed Biotin-Operated Ligand Trapping (BOLT), which combines a HaloTag-fused POI with a streptavidin trap. Desthiobiotin-conjugated Janelia Fluor HaloTag ligands enable POI visualization, trapping, and biotin-operated release. We demonstrate BOLT's capacity in synchronized secretory cargo release, microtubule-directed organelle movement and hitchhiking, and ectopically localizing nuclear transcription factor. Importantly, pulse-chase BOLT further reveals bidirectional endoplasmic reticulum-Golgi trafficking of an endogenous Golgi enzyme and distinguishes recycled from newly synthesized enzyme populations using distinct HTLs. Altogether, BOLT provides an all-in-one platform to interrogate how subcellular localization regulates protein function across HaloTag-compatible systems.

synthetic biology↗

bioRxiv2026

LRRC57/RABIN is a presynaptic inhibitor of Rab GTPases in glutamatergic neurons

Synaptic vesicle cycling, if not properly constrained, can result in excessive neurotransmitter release and subsequent neural pathology. Rab GTPases orchestrate synaptic vesicle trafficking through GTP-dependent interactions with effector proteins, but the restraining mechanism of these interactions is unknown. Here we identify LRRC57 (or RABIN for RAB INhibitor), a conserved brain-enriched protein in glutamatergic synapses that binds multiple GTP-loaded synaptic Rabs and competitively blocks access to their effectors. Loss of Rabin increased glutamate release, expanded vesicle pools, accelerated vesicle turnover, and produced circuit hyperexcitability with epileptiform activity, which was mitigated by an antiepileptic agent that targets presynaptic function. Conversely, overexpression of the Rabin gene suppressed neurotransmitter release and protected against induced seizures and persistent epileptiform discharges. Together, these findings define a noncanonical decoy-effector mechanism that constrains presynaptic Rab signaling to preserve excitatory circuit stability.

neuroscience↗

bioRxiv2026

MYC/MIZ1 suppression of lysosomal protein degradation drives immune evasion in pancreatic ductal adenocarcinoma

The MYC oncoprotein promotes immune evasion of pancreatic ductal adenocarcinoma (PDAC), but the underlying molecular mechanisms are not fully understood. Here we show that MYC protects PDAC tumors from CD4+ T cell-dependent elimination. Single cell sequencing shows that MYC suppression in tumor cells increases amino acid availability and broadly activates amino acid-responsive gene expression programs in immune cell populations. This occurs because MYC-driven uptake depletes free amino acids from tumor interstitial fluid and plasma, while MYC compromises macropinocytosis and autophagy, both of which depend on lysosomal protein degradation. MYC engages the POZ/BTB transcription factor MIZ1 to suppress lysosomal genes regulated by the TFE3/TFEB/MITF network or by free MIZ1, thereby inhibiting lysosomal protein degradation. An orthogonal genetic model enabling transient, selective inhibition of amino acid uptake in tumor cells recapitulates the effects of MYC depletion on amino acid levels in the tumor microenvironment and induces complete, CD4+ T cell-dependent tumor eradication with long-term survival. We propose that MYC-mediated, cell-autonomous disruption of lysosome function coupled to non-cell-autonomous protection from immune clearance allows MYC-low cells to benefit from MYC-high neighbors, such that intratumoral heterogeneity in MYC expression confers a selective advantage to the entire tumor. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/740267v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@140d2ccorg.highwire.dtl.DTLVardef@cf5f1borg.highwire.dtl.DTLVardef@6d132aorg.highwire.dtl.DTLVardef@10535e6_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Alternative organelle targeting of OPA1 mediates fatty acid release from lipid droplets

Mitochondria and lipid droplets (LDs) are functionally coupled to coordinate fatty acid utilization and storage. However, a comprehensive understanding of mitochondria-LD alliances remains elusive. We have identified a previously unrecognized role for optical atrophy 1 (OPA1), a mitochondrial fusion factor, in the regulation of fatty acid release from LDs. We demonstrated that OPA1s exon 4 adapts an amphipathic helix to target OPA1 to LDs. OPA1 localized to LDs promote fatty acid release by facilitating the recruitment of lipases to LDs. In addition, OPA1s residence on LDs competes with its mitochondrial entry, influencing mitochondria fusion and connectivity. Furthermore, the S158N polymorphism within OPA1s exon 4 exhibiting attenuated fatty acid release from LDs is associated with changes in metabolic traits in pediatric cancer survivors. Altogether, our findings reveal that OPA1 actively mediates fatty acid release from LDs and provide a mechanistic link between OPA1 and human metabolism.

cell biology↗

bioRxiv2026

Elevated endocytic trafficking mediated by GPRASP2 maintains HSC fidelity.

Endolysosomal trafficking supports cellular homeostasis through coordinated regulation of extrinsic signaling inputs. Hematopoietic stem cell (HSC) function requires a tightly regulated balance between quiescence for long-term preservation and rapid activation for blood production. Although lysosomal regulation of metabolism and quiescence has been linked to stem cell maintenance, the contribution of endocytic routing to lysosomal function remains incompletely understood. Here, we show that quiescent HSCs rely on elevated endocytosis to maintain self-renewal. This activity is mediated in part by GPRASP2, a post-endosomal sorting protein. HSCs enriched in GPRASP2 are functionally dormant yet molecularly primed for activation however, in response to proliferative cues, dormant HSCs exhibit reduced signaling and proliferation. Disruption of GPRASP2-mediated endocytosis induces rapid proliferation and increased expression of signaling constituents, consistent with a model in which elevated endocytosis attenuates signaling through receptor internalization. Thus, we identify endocytosis as a mechanism by which HSCs limit functional exhaustion arising from chronic activation.

cell biology↗

bioRxiv2025

ER-to-Golgi Trafficking is a Nutrient-Sensitive Checkpoint Linking Glucose Starvation to Cell Surface Remodeling

Cancer cells adapt to nutrient stress by remodeling the repertoire of proteins on their surface, enabling survival and progression under starvation conditions. However, the molecular mechanisms by which nutrient cues reshape the cell surface proteome to influence cell behavior remain largely unresolved. Here, we show that acute glucose starvation, but not amino acid deprivation or mTOR inhibition, selectively impairs ER-to-Golgi export of specific cargoes, such as E-cadherin, in a SEC24C-dependent manner. Quantitative cell surface proteomics reveal that glucose deprivation remodels the cell surface proteome, notably reducing surface expression of key adhesion molecules. This nutrient-sensitive reprogramming enhances cell migration in vitro and promotes metastasis in vivo. Mechanistically, we show that AMPK and ULK1 signaling orchestrate this process independent of autophagy, with ULK1-mediated phosphorylation of SEC31A driving SEC24C-dependent COPII reorganization. These findings establish ER-to-Golgi trafficking as a nutrient-sensitive regulatory node that links metabolic stress to cell surface remodeling and metastatic potential.

cell biology↗

bioRxiv2025

Deciphering acquired resistance mechanisms to sustained auxin-inducible protein degradation in cells and mice

Targeted protein degradation is a favorable strategy for studying the immediate downstream effects of protein loss-of-function. An appealing platform among these technologies is the auxin-inducible degron (AID) system. Although this system has been applied extensively to cell and animal models, degradation resistance to long-term auxin treatment has not been studied. With the advent of the new AID2 system, cellular toxicity due to the high concentrations of auxin required in the original AID1 system is no longer a concern, making it possible to study protein degradation over extended periods. In this study, we derived multiple miniAID-tagged knock-in human cell lines and a Ctcf-miniAID knock-in mouse strain to investigate mechanisms of degradation resistance. We revealed four independent resistance mechanisms, including a nonsense mutation in the CTCF coding sequence that removed the miniAID peptide, a missense point mutation in the miniAID coding region that disrupted ubiquitin complex targeting, and silencing of the OsTIR1 adaptor protein. Resistance to auxin degradation was also acquired in mouse primary CtcfminiAID/miniAID knock-in B-ALL cells through missense mutations of the OsTIR1(F74G) protein in vivo and ex vivo. In summary, our innovative study expands our understanding of the AID system and cautions careful consideration of design for future applications in mammalian system.

cell biology↗

bioRxiv2025

In vitro plasticity between ureteric epithelial and distal nephron identity and maturity is controlled by extracellular signals.

Several studies have described human pluripotent stem cell (hPSC)-derived ureteric epithelium, which in the embryo arises from the nephric duct and forms the collecting ducts of the kidney. However, hPSC-derived distal nephron epithelium can also adopt a ureteric phenotype, despite this not occurring during embryogenesis. In this study, RETtdTomato and GATA3mCherry reporter lines were used to further investigate this plasticity. Induction of anterior intermediate mesoderm resulted in the spontaneous formation of an epithelial plexus with a nephric duct-like identity. Subsequent addition of RSPO1 induced patterning of distalized nephrons, including distal convoluted tubule and thick ascending limb of loop of Henle but lacking proximal segments or glomeruli. This epithelium showed a capacity to adopt ureteric epithelial or nephric duct-like states in ex vivo co-culture in response to external cues. The same epithelium seeded as single cells in Matrigel formed epithelial spheroids and adopted a RET+ ureteric tip identity. This in vitro continuum between nephric duct, ureteric epithelium and distal nephron illustrates the role of the microenvironment in cellular identity. Highlights and eTOCO_ST_ABSHighlightsC_ST_ABSO_LIDistal renal epithelium shows substantial plasticity in vitro C_LIO_LIIdentity can be shifted towards distal nephron, including distal convoluted tubule and loop of Henle, in response to prolonged RSPO1 C_LIO_LIDistal epithelial identity can shift in ex vivo culture in response to surrounding signals C_LIO_LICollecting duct is supported by Matrigel culture C_LI eTOC blurbKairath and colleagues investigate the plasticity of induced renal epithelium in response to external signalling cues in vitro and ex vivo. Initial patterning generates a plastic epithelium able to pattern to specific distal nephron. The same epithelium, cultured in Matrigel patterns to ureteric epithelium.

developmental biology↗

bioRxiv2025

Targeting Regnase-1 unleashes CAR T cell antitumor activity for osteosarcoma and creates a proinflammatory tumor microenvironment

Negative regulators of T cell function represent promising targets to enhance the intrinsic antitumor activity of CAR T cells against solid tumors. However, the endogenous immune ecosystem in solid tumors often represents an immunosuppressive therapeutic barrier to CAR T cell therapy, and it is currently unknown whether deletion of negative regulators in CAR T cells reshapes the endogenous immune landscape. To address this knowledge gap, we developed CAR T cells targeting B7-H3 in immune-competent osteosarcoma models and evaluated the intrinsic and extrinsic effects of deleting a potent negative regulator called Regnase-1 (Reg-1). Deletion of Reg-1 not only improved the effector function of B7-H3-CAR T cells but also endowed them with the ability to create a proinflammatory landscape characterized by an influx of IFN{gamma}-producing endogenous T cells and NK cells and a reduction of inhibitory myeloid cells, including M2 macrophages. Thus, deleting negative regulators in CAR T cells enforces a non-cell-autonomous state by creating a proinflammatory tumor microenvironment.

immunology↗

bioRxiv2025

Midkine Attenuates Aβ Fibril Assembly and Amyloid Plaque Formation

Proteomic profiling of Alzheimers disease (AD) brains has identified numerous understudied proteins, including midkine (MDK), that are highly upregulated and correlated with A{beta} since the early disease stage, but their roles in disease progression are not fully understood. Here we present that MDK attenuates A{beta} assembly and influences amyloid formation in the 5xFAD amyloidosis mouse model. MDK protein mitigates fibril formation of both A{beta}40 and A{beta}42 peptides in Thioflavin T fluorescence assay, circular dichroism, negative stain electron microscopy, and NMR analysis. Knockout of Mdk gene in 5xFAD increases amyloid formation and microglial activation. Further comprehensive mass spectrometry-based profiling of whole proteome and detergent-insoluble proteome in these mouse models indicates significant accumulation of A{beta} and A{beta}-correlated proteins, along with microglial components. Thus, our structural and mouse model studies reveal a protective role of MDK in counteracting amyloid pathology in Alzheimers disease.

biochemistry↗

bioRxiv2025

Granular component sub-phases direct ribosome biogenesis in the nucleolus

The hierarchical, multiphase organization of the nucleolus underlies ribosome biogenesis. Ribonucleoprotein particles that regulate ribosomal subunit assembly are heterogeneously disposed in the granular component (GC) of the nucleolus. However, the molecular origins of the GCs spatial heterogeneity and its association with ribosomal subunit assembly remain poorly understood. Here, using super-resolution microscopy, we uncover that key GC biomolecules, including nucleophosmin (NPM1), surfeit locus protein 6 (SURF6), and ribosomal RNA (rRNA), are heterogeneously localized within sub-phases in the GC. In vitro reconstitution showed that these GC biomolecules form multiphase condensates with SURF6/rRNA-rich core and NPM1-rich shell, providing a mechanistic basis for GCs spatial heterogeneity. SURF6s association with rRNA is weakened upon ribosome subunit assembly, enabling NPM1 to extract assembled subunits from condensates--suggesting an assembly-line-like mechanism of subunit efflux from the GC. Our results establish a framework for understanding the heterogeneous structure of the GC and reveal how its distinct sub-phases facilitate ribosome subunit assembly.

biochemistry↗

bioRxiv2025

Receptor Allostery Promotes Context-Specific Sonic Hedgehog Signaling During Embryonic Development

Sonic Hedgehog (SHH) signaling functions in temporal- and context-dependent manners to pattern diverse tissues during embryogenesis. The signal transducer Smoothened (SMO) is activated by sterols, oxysterols, and arachidonic acid (AA) through binding pockets in its extracellular cysteine-rich domain (CRD) and 7-transmembrane (7TM) bundle. In vitro analyses suggest SMO signaling is allosterically enhanced by combinatorial ligand binding to these pockets but in vivo evidence of SMO allostery is lacking. Herein, we map an AA binding pocket at the top of the 7TM bundle and show that its disruption attenuates SHH and sterol-stimulated SMO induction. A knockin mouse model of compromised AA binding reveals that homozygous mutant mice are cyanotic, exhibit high perinatal lethality, and show congenital heart disease. Surviving mutants demonstrate pulmonary maldevelopment and fail to thrive. Neurodevelopment is unaltered in these mice, suggesting that context-dependent allosteric regulation of SMO signaling allows for precise tuning of pathway activity during cardiopulmonary development.

developmental biology↗

bioRxiv2024

Spatially Resolved in vivo CRISPR Screen Sequencing via Perturb-DBiT

Perturb-seq enabled the profiling of transcriptional effects of genetic perturbations in single cells but lacks the ability to examine the impact on tissue environments. We present Perturb-DBiT for simultaneous co- sequencing of spatial transcriptome and guide RNAs (gRNAs) on the same tissue section for in vivo CRISPR screen with genome-scale gRNA libraries, offering a comprehensive understanding of how genetic modifications affect cellular behavior and tissue architecture. This platform supports a variety of delivery vectors, gRNA library sizes, and tissue preparations, along with two distinct gRNA capture methods, making it adaptable to a wide range of experimental setups. In applying Perturb-DBiT, we conducted un-biased knockouts of tens of genes or at genome-wide scale across three cancer models. We mapped all gRNAs in individual colonies and corresponding transcriptomes in a human cancer metastatic colonization model, revealing clonal dynamics and cooperation. We also examined the effect of genetic perturbation on the tumor immune microenvironment in an immune-competent syngeneic model, uncovering differential and synergistic perturbations in promoting immune infiltration or suppression in tumors. Perturb-DBiT allows for simultaneously evaluating the impact of each knockout on tumor initiation, development, metastasis, histopathology, and immune landscape. Ultimately, it not only broadens the scope of genetic inquiry, but also lays the groundwork for developing targeted therapeutic strategies.

genomics↗

bioRxiv2024

Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors

BackgroundMedulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. MethodsTo determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Childrens Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. ResultsOur RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. ConclusionsThese results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key PointsO_LISeveral Type I MAGE CTAs are expressed in >60% of group 3 MBs. C_LIO_LIType I MAGEs affect MB cell proliferation and apoptosis. C_LIO_LIMAGEs are potential biomarkers and therapeutic targets for group 3 MBs. C_LI Importance of the StudyThis study is the first comprehensive analysis of all Type I MAGE CTAs (MAGEA, -B, and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/594201v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1ac274aorg.highwire.dtl.DTLVardef@1479944org.highwire.dtl.DTLVardef@19f5aaforg.highwire.dtl.DTLVardef@fad069_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy--a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RAs overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RAs ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.

cancer biology↗

bioRxiv2024

Synaptic plasticity in human thalamocortical assembloids

Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA-sequencing revealed that most cells in mature thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits. HighlightsO_LIHuman thalamic organoids consist of mostly glutamatergic projection neurons. C_LIO_LIThalamocortical assembloids form reciprocal glutamatergic synapses. C_LIO_LISynapses are functional and undergo short-term plasticity resembling animal models. C_LIO_LILong-term potentiation and depression reveal mechanisms distinct from rodents. C_LI eTOCHuman organoids are often used to model diseases with synaptic pathology; however, few studies have examined synaptic function via single-cell or single-synapse recordings. Patton et al. fused human thalamic and cortical organoids into assembloids to examine synaptic transmission and short- and long-term synaptic plasticity in human thalamocortical and corticothalamic circuits. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/578421v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@21546org.highwire.dtl.DTLVardef@c2ba28org.highwire.dtl.DTLVardef@4bc28aorg.highwire.dtl.DTLVardef@1d12a98_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Patient derived model of UBA5-associated encephalopathy identifies defects in neurodevelopment and highlights potential therapies

UBA5 encodes for the E1 enzyme of the UFMylation cascade, which plays an essential role in ER homeostasis. The clinical phenotypes of UBA5-associated encephalopathy include developmental delays, epilepsy and intellectual disability. To date, there is no humanized neuronal model to study the cellular and molecular consequences of UBA5 pathogenic variants. We developed and characterized patient-derived cortical organoid cultures and identified defects in GABAergic interneuron development. We demonstrated aberrant neuronal firing and microcephaly phenotypes in patient-derived organoids. Mechanistically, we show that ER homeostasis is perturbed along with exacerbated unfolded protein response pathway in cells and organoids expressing UBA5 pathogenic variants. We also assessed two gene expression modalities that augmented UBA5 expression to rescue aberrant molecular and cellular phenotypes. Our study provides a novel humanized model that allows further investigations of UBA5 variants in the brain and highlights novel systemic approaches to alleviate cellular aberrations for this rare, developmental disorder. One-sentence summaryPatient derived model of UBA5-assoicated DEE recapitulated disease phenotype, revealed defects in neurodevelopment, and highlighted potential therapies.

genetics↗