Abstract Title

A High Throughput and Integrative Approach to Evaluating the Functional Significance of a Glycosidase NGLY1 in Human Brain Development

Presenter Name

Victor Lin

RAD Assignment Number

1807

Abstract

Background: Mutations of the NGLY1 gene, leading to NGLY1 deficiency and associated neurodysfunction in pediatric patients, have been identified as the cause of a previously undiagnosed congenital disorder of deglycosylation. Despite the identification of the causal mutations, how NGLY1 deficiency disturbs normal cerebral development and causes neurological abnormalities is unknown.

Purpose: Our desire is to unravel the mystery behind this novel disease and how it influences the assembly and function of the human cerebral landscape. Further, our hope is to develop mid-to-high throughput platforms that can be applied to discover and test druggable targets for this disease and adapted for associated neurocognitive or neurodegenerative disorders.

Methods: Using human induced pluripotent stem cells (hiPSCs) and the state-of-the-art gene editing technology, CRISPR-Cas9, NGLY1 deficient human pluripotent stem cells (hPSCs) were created and used to elucidate the disease pathophysiology. In succession, middle-to-high throughput platforms were applied to recapitulate the disease in 2D and 3D, used in tandem with systems biology and novel imaging capabilities to discover new understandings and the importance glycosylation states for cerebral development and function.

Results: The CRISPR-Cas9 mediated knockout of NGLY1 was confirmed by DNA sequencing and a biochemical test. Our optimized two-dimensional and three-dimensional differentiation protocols for neurogenesis in the control and NGLY1-deficient hESCs and hiPSCs showed that the loss of NGLY1 appears to have a negligible impact on the viability and cellular pluripotency in undifferentiated hPSCs. Neuroepithelial differentiation can be successfully generated in both control and NGLY1-deficient hPSCs, suggesting that the commitment of hPSCs to the neural lineage is not profoundly hindered by the loss of NGLY1 activity. However, compared with the differentiated derivatives of control hPSCs, the derivatives from neural differentiation in NGLY1-deficient hPSCs showed noticeably increased apoptosis, suggesting that NGLY1 activity may play a critical role in the viability of neural progenitor cells, as well as, play a role in the success of their subsequent differentiation into neuronal or astroglial lineages.

Conclusions: We have built a new and unique model that can recapitulate the early-stage neurodevelopment patterns associated with NGLY1 deficiency. Using systems biology and imaging approaches, we are uncovering unprecedented insights into this newly identified disease. With the hiPSC and CRISPR-Cas9 gene editing, we demonstrate how regenerative medicine and genetic engineering approaches can be applied to studying the pathogenesis of human hereditary disease, applied in like to understand other brain pathologies, and possibly assist in the discovery of new therapeutics.

Research Area

Neuroscience

Presentation Type

Poster

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A High Throughput and Integrative Approach to Evaluating the Functional Significance of a Glycosidase NGLY1 in Human Brain Development

Background: Mutations of the NGLY1 gene, leading to NGLY1 deficiency and associated neurodysfunction in pediatric patients, have been identified as the cause of a previously undiagnosed congenital disorder of deglycosylation. Despite the identification of the causal mutations, how NGLY1 deficiency disturbs normal cerebral development and causes neurological abnormalities is unknown.

Purpose: Our desire is to unravel the mystery behind this novel disease and how it influences the assembly and function of the human cerebral landscape. Further, our hope is to develop mid-to-high throughput platforms that can be applied to discover and test druggable targets for this disease and adapted for associated neurocognitive or neurodegenerative disorders.

Methods: Using human induced pluripotent stem cells (hiPSCs) and the state-of-the-art gene editing technology, CRISPR-Cas9, NGLY1 deficient human pluripotent stem cells (hPSCs) were created and used to elucidate the disease pathophysiology. In succession, middle-to-high throughput platforms were applied to recapitulate the disease in 2D and 3D, used in tandem with systems biology and novel imaging capabilities to discover new understandings and the importance glycosylation states for cerebral development and function.

Results: The CRISPR-Cas9 mediated knockout of NGLY1 was confirmed by DNA sequencing and a biochemical test. Our optimized two-dimensional and three-dimensional differentiation protocols for neurogenesis in the control and NGLY1-deficient hESCs and hiPSCs showed that the loss of NGLY1 appears to have a negligible impact on the viability and cellular pluripotency in undifferentiated hPSCs. Neuroepithelial differentiation can be successfully generated in both control and NGLY1-deficient hPSCs, suggesting that the commitment of hPSCs to the neural lineage is not profoundly hindered by the loss of NGLY1 activity. However, compared with the differentiated derivatives of control hPSCs, the derivatives from neural differentiation in NGLY1-deficient hPSCs showed noticeably increased apoptosis, suggesting that NGLY1 activity may play a critical role in the viability of neural progenitor cells, as well as, play a role in the success of their subsequent differentiation into neuronal or astroglial lineages.

Conclusions: We have built a new and unique model that can recapitulate the early-stage neurodevelopment patterns associated with NGLY1 deficiency. Using systems biology and imaging approaches, we are uncovering unprecedented insights into this newly identified disease. With the hiPSC and CRISPR-Cas9 gene editing, we demonstrate how regenerative medicine and genetic engineering approaches can be applied to studying the pathogenesis of human hereditary disease, applied in like to understand other brain pathologies, and possibly assist in the discovery of new therapeutics.