Deficiency of N-glycanase 1 perturbs neurogenesis and cerebral development modeled by human organoids.

Mutations in N-glycanase 1 (NGLY1), which deglycosylates misfolded glycoproteins for degradation, can cause NGLY1 deficiency in patients and their abnormal fetal development in multiple organs, including microcephaly and other neurological disorders. Using cerebral organoids (COs) developed from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), we investigate how NGLY1 dysfunction disturbs early brain development. While NGLY1 loss had limited impact on the undifferentiated cells, COs developed from NGLY1-deficient hESCs showed defective formation of SATB2-positive upper-layer neurons, and attenuation of STAT3 and HES1 signaling critical for sustaining radial glia. Bulk and single-cell transcriptomic analysis revealed premature neuronal differentiation accompanied by downregulation of secreted and transcription factors, including TTR, IGFBP2, and ID4 in NGLY1-deficient COs. NGLY1 malfunction also dysregulated ID4 and enhanced neuronal differentiation in CO transplants developed in vivo. NGLY1-deficient CO cells were more vulnerable to multiple stressors; treating the deficient cells with recombinant TTR reduced their susceptibility to stress from proteasome inactivation, likely through LRP2-mediated activation of MAPK signaling. Expressing NGLY1 led to IGFBP2 and ID4 upregulation in CO cells developed from NGLY1-deficiency patient's hiPSCs. In addition, treatment with recombinant IGFBP2 enhanced ID4 expression, STAT3 signaling, and proliferation of NGLY1-deficient CO cells. Overall, our discoveries suggest that dysregulation of stress responses and neural precursor differentiation underlies the brain abnormalities observed in NGLY1-deficient individuals.

Urine Sample-Derived Cerebral Organoids Suitable for Studying Neurodevelopment and Pharmacological Responses.

Cerebral organoids (COs) developed from human induced pluripotent stem cells (hiPSCs) have been noticed for their potential in research and clinical applications. While skin fibroblast-derived hiPSCs are proficient at forming COs, the cellular and molecular features of COs developed using hiPSCs generated from other somatic cells have not been systematically examined. Urinary epithelial cells (UECs) isolated from human urine samples are somatic cells that can be non-invasively collected from most individuals. In this work, we streamlined the production of COs using hiPSCs reprogrammed from urine sample-derived UECs. UEC-derived hiPSC-developed COs presented a robust capacity for neurogenesis and astrogliogenesis. Although UEC-derived hiPSCs required specific protocol optimization to properly form COs, the cellular and transcriptomic features of COs developed from UEC-derived hiPSCs were comparable to those of COs developed from embryonic stem cells. UEC-derived hiPSC-developed COs that were initially committed to forebrain development showed cellular plasticity to transition between prosencephalic and rhombencephalic fates in vitro and in vivo, indicating their potential to develop into the cell components of various brain regions. The opposite regulation of AKT activity and neural differentiation was found in these COs treated with AKT and PTEN inhibitors. Overall, our data reveal the suitability, advantage, and possible limitations of human urine sample-derived COs for studying neurodevelopment and pharmacological responses.

Bioinspired Nanoparticles Engineered for Enhanced Delivery to the Bone.

Targeting therapeutic agents to specific organs in the body remains a challenge despite advances in the science of systemic drug delivery. We have engineered a programmable-bioinspired nanoparticle (P-BiNP) delivery system to simultaneously target the bone and increase uptake in homotypic tumor cells by coating polymeric nanoparticles with programmed cancer cell membranes. This approach is unique in that we have incorporated relevant clinical bioinformatics data to guide the design and enhancement of biological processes that these nanoparticles are engineered to mimic. To achieve this, an analysis of RNA expression from metastatic prostate cancer patients identified ITGB3 (a subunit of integrin α V ß 3) as overexpressed in patients with bone metastasis. Cancer cells were stimulated to increase this integrin expression on the cell surface, and these membranes were subsequently used to coat cargo carrying polymeric nanoparticles. Physicochemical optimization and characterization of the P-BiNPs showed desirable qualities regarding size, ζ potential, and stability. In vitro testing confirmed enhanced homotypic binding and uptake in cancer cells. P-BiNPs also demonstrated improved bone localization in vivo with a murine model. This novel approach of identifying clinically relevant targets for dual homotypic and bone targeting has potential as a strategy for treatment and imaging modalities in diseases that affect the bone as well as broader implications for delivering nanoparticles to other organs of interest.

Stress and interferon signalling-mediated apoptosis contributes to pleiotropic anticancer responses induced by targeting NGLY1.

BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.

Potassium as a pluripotency-associated element identified through inorganic element profiling in human pluripotent stem cells.

Despite their well-known function in maintaining normal cell physiology, how inorganic elements are relevant to cellular pluripotency and differentiation in human pluripotent stem cells (hPSCs) has yet to be systematically explored. Using total reflection X-ray fluorescence (TXRF) spectrometry and inductively coupled plasma mass spectrometry (ICP-MS), we analyzed the inorganic components of human cells with isogenic backgrounds in distinct states of cellular pluripotency. The elemental profiles revealed that the potassium content of human cells significantly differs when their cellular pluripotency changes. Pharmacological treatment that alters cell membrane permeability to potassium affected the maintenance and establishment of cellular pluripotency via multiple mechanisms in bona fide hPSCs and reprogrammed cells. Collectively, we report that potassium is a pluripotency-associated inorganic element in human cells and provide novel insights into the manipulation of cellular pluripotency in hPSCs by regulating intracellular potassium.