<i>Sod1</i> trisomy causes ENS developmental defects and susceptibility to Hirschsprung disease via neuronal <i>Ret</i> suppression and glial remodeling

Down syndrome (DS; trisomy 21) confers a ~100-fold increased risk of Hirschsprung disease (HSCR), yet the causal contributions of specific chromosome 21 genes remain unresolved. Here we show that increased dosage of SOD1 alone is sufficient to perturb enteric nervous system (ENS) development. We engineered a humanized <i>SOD1</i> trisomic mouse line by inserting a 28 kb human <i>SOD1</i> locus into ROSA26 and genetically profiled the distal colon at postnatal day 0 using single-cell RNA-seq and immunofluorescence. <i>Sod1/SOD1</i> was elevated ~1.5X in the ENS but varied by cell type, with transcriptionally active progenitors showing the greatest increase. Cell composition shifted toward transcriptionally active cells and glia, with concomitant loss of excitatory and inhibitory motor neurons and interneurons. Genetically, <i>Sod1/SOD1</i> trisomy downregulated synaptic and neuronal communication programs but upregulated DNA replication/cell-cycle and genome maintenance pathways, especially within glia. Consequently, key HSCR genes were dysregulated: <i>Ret</i>, <i>Ednrb</i>, and <i>Sema3c</i> were decreased, while <i>Sema3a</i> ,a negative guidance cue, was increased. <i>Ret</i> was selectively reduced in inhibitory and excitatory motor neurons and progenitors, unchanged in glia, and reduced at the protein level <i>in vivo</i>. Within glia, Sod1/SOD1 was particularly elevated in proliferating/active glia with a glia-specific bias toward endogenous mouse <i>Sod1</i> expression. Taken together, these data support a dual mechanism whereby increased <i>Sod1/SOD1</i> dosage suppresses <i>RET</i>-dependent neurogenesis while independently promoting reactive/proliferative glial states. Thus, <i>SOD1</i> is sufficient to alter ENS development significantly and provide the susceptibility substrate for HSCR with further reductions in <i>RET</i> gene expression leading to aganglionosis.