NAD+ metabolic collapse and arrest of epithelial development in Hirschsprung disease

Hirschsprung disease (HSCR) is characterized by distal aganglionosis. Persistent postoperative dysmotility and enterocolitis suggest that intrinsic epithelial vulnerabilities may remain within the ganglionic bowel. We here aimed to define both the state of the epithelium and its metabolism in HSCR.

Distal colonic tissues from patients with HSCR and non-HSCR controls (HSCR allied disorders) were examined by single-cell RNA sequencing. Particular attention was given to nicotinamide adenine dinucleotide (NAD⁺) metabolism and oxidative phosphorylation across epithelial subsets. Immunofluorescence and immunohistochemistry for γH2A.X, PARP1, TOM20, KRT20, and poly(ADP-ribose) (PAR) validated transcriptional signatures of cellular stress, metabolic remodeling, and impaired epithelial maturation.

Compared with control, HSCR epithelium showed marked transcriptional remodeling, with accumulation of stem and transit-amplifying cells, and depletion of differentiated epithelial lineages, indicating impaired epithelial maturation. These changes were most pronounced in the distal ganglionic colon with a reduced epithelial KRT20 signal, where oxidative phosphorylation, ATP synthesis, and lipid and steroid metabolic pathways were broadly suppressed. Key NAD⁺ biosynthetic enzymes were downregulated, while NAD⁺-consuming sirtuins and PAR polymerases (PARP) were upregulated, particularly in HSCR ganglionic colon crypt populations. Similarly, HSCR tissues exhibited increased crypt γH2A.X and nuclear PARP1, as well as elevated TOM20 and poly/mono-ADP-ribose staining, consistent with metabolic and genomic stress.

HSCR epithelium displays metabolic fragility characterized by NAD⁺ biosynthetic insufficiency, increased NAD⁺ consumption, mitochondrial remodeling, DNA damage, and impaired epithelial maturation. These epithelial alterations complement enteric nervous system (ENS)-centered disease models and suggest that NAD⁺-related pathways may represent modifiable programs relevant to postoperative mucosal resilience and susceptibility to Hirschsprung disease-associated enterocolitis (HAEC).