NGLY1 deficiency is a rare, congenital disorder that is caused by mutations in the cytoplasmic peptide:N- glycanase, an enzyme associated with degradation of misfolded proteins. Symptoms of the disease include neuropathy with movement disorder, global developmental delay, liver damage, chronic constipation and the absence of tears. As NGLY1 deficiency has only recently been identified through whole exome sequencing of patients, no targeted therapies are available. Investigations into NGLY1 expression and function throughout the body are therefore imperative for developing better strategies for clinical management of this condition. The central hypothesis of this research proposal is loss of NGLY1 function elicits an ER stress response in the intestine that alters epithelial differentiation while abrogating enteric nervous system function, changes which together result in intestinal dysmotility. We have based this hypothesis on the key clinical features in patients and preliminary studies using human intestinal organoids (HIOs), or ?mini-guts?, generated by differentiation of control (hESC) and patient-derived pluripotent stem cell (iPS) lines. HIOs generated from NGLY1-/- iPSCs, when transplanted into immunocompromised mice, form mature intestinal tissue which undergoes differentiation and morphogenesis to form intestinal villi, but with epithelial secretory cell hyperplasia (Goblet and enteroendocrine cells). Therefore, in Specific Aim 1a I will characterize the phenotype and function of control iPSC-derived HIOs (NGLY1+/+) compared to HIOs generated from iPSC lines established from the fibroblasts of NGLY1 deficient patients (NGLY1-/-) and their parents (NGLY1+/-). Intestinal cell lineages will be quantified through immunostaining and transplanted HIOs expanded ex vivo as human intestinal enteroid (HIE) monolayers to assess intestinal barrier function, secretion and absorption.
Specific Aim 1 b will complement these studies by quantifying ER stress responses within these HIOs, as NGLY1 associates with the endoplasmic reticulum associated degradation (ERAD) machinery. Collectively, we expect to confirm our initial finding of increases in secretory cell populations and identify that secretory cell function may be inefficient due to elevated ER stress and the inhibition of protein translation. As chronic constipation is a debilitating symptom of NGLY1 deficiency but the contribution of NGLY1 to intestinal motility is unknown, in Specific Aim 2 we will incorporate enteric neurons into HIOs, representing each NGLY1 genotype in each compartment (neuronal or epithelial), to assess their function and interaction with the muscle layer. To do this, we will work with collaborators who have demonstrated that a functional enteric neural network develops in xenografted HIOs when they are combined with neural crest progenitors before transplantation (HIOs + ENS). Immunostaining will be used to assess neuronal differentiation with analysis of contractility using isometric force assays in organ chambers. At the completion of these studies we expect to have elucidated how mutations in NGLY1 impact intestinal differentiation and function.

Public Health Relevance

NGLY1 deficiency is a rare genetic disorder which falls under the spectrum of conditions classified as congenital disorders of glycosylation. The development of a human intestinal organoid (HIO) system to model the gastrointestinal characteristics of this disease may prove essential in unraveling how mutations in NGLY1 impair intestinal motility. It is also likely that the results of the proposed investigation will contribute new information to our understanding of how cell stress modulates intestinal homeostasis and differentiation with relevance for multiple gastrointestinal disorders, such as inflammatory bowel disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32DK112511-01
Application #
9256798
Study Section
Special Emphasis Panel (ZDK1)
Program Officer
Densmore, Christine L
Project Start
2017-06-19
Project End
2019-06-18
Budget Start
2017-06-19
Budget End
2018-06-18
Support Year
1
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
051113330
City
Houston
State
TX
Country
United States
Zip Code
77030