Vitamin B12 (cobalamin) is a key determinant of S-adenosyl methionine (SAM)-dependent epigenomic cellular regulations related to methylation/acetylation and its deficiency produces neurodegenerative disorders by elusive mechanisms. Sirtuin 1 deacetylase (SIRT1) triggers cell response to nutritional stress through endoplasmic reticulum (ER) stress. Recently, we have established a N1E115 dopaminergic cell model by stable expression of a transcobalamin–oleosin chimera (TO), which impairs cellular availability of vitamin B12, decreases methionine synthase activity and SAM level, and reduces cell proliferation. In contrast, oleosin-transcobalamin chimera (OT) does not modify the phenotype of transfected cells. Presently, the impaired cellular availability of vitamin B12 in TO cells activated irreversible ER stress pathways, with increased P-eIF-2α, P-PERK, P-IRE1α, ATF6, ATF4, decreased chaperon proteins and increased pro-apoptotic markers, CHOP and cleaved caspase 3, through reduced SIRT1 expression and consequently greater acetylation of heat-shock factor protein 1 (HSF1). Adding either B12, SIRT1, or HSF1 activators as well as overexpressing SIRT1 or HSF1 dramatically reduced the activation of ER stress pathways in TO cells. Conversely, impairing SIRT1 and HSF1 by siRNA, expressing a dominant negative form of HSF1, or adding a SIRT1 inhibitor led to B12-dependent ER stress in OT cells. Addition of B12 abolished the activation of stress transducers and apoptosis, and increased the expression of protein chaperons in OT cells subjected to thapsigargin, a strong ER stress stimulator. AdoX, an inhibitor of methyltransferase activities, produced similar effects than decreased B12 availability on SIRT1 and ER stress by a mechanism related to increased expression of hypermethylated in cancer 1 (HIC1). Taken together, these data show that cellular vitamin B12 has a strong modulating influence on ER stress in N1E115 dopaminergic cells. The impaired cellular availability in vitamin B12 induces irreversible ER stress by greater acetylation of HSF1 through decreased SIRT1 expression, whereas adding vitamin B12 produces protective effects in cells subjected to ER stress stimulation.

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Background and Hypothesis: Congenital heart disease(CHD) is the most common birth defect, but most genetic contributors remain unknown. We recently identified CHD patients with variants in a gene called SHROOM3. The SHROOM3 protein impacts the actin cytoskeleton by binding ActinF and Rho-kinase, causing actomyosin constriction. SHROOM3 also binds Dishevelled2(Dvl2), a component of Wnt/Planar cell polarity(PCP) signaling pathway, suggesting a connection between PCP signaling and actin-myosin contraction. We hypothesize SHROOM3 disruption alters PCP signaling and actin cytoskeleton during cardiac development, and is a novel contributor to CHD.   Project Methods: We analyzed the cardiac phenotype of Shroom3 gene trap knockout mice at embryonic day 14.5. We characterized the expression of Shroom3 during cardiac development using LacZ staining at important stages of cardiac development. Using IHC, we measured actomyosin disruption in Shroom3 knockout embryos. We preformed in silico analysis on previously identified SHROOM3 variants from patients with CHD.   Results: Shroom3 null mice had Ventricular Septal Defects (0.73, p=0.0006), Double Outlet Right Ventricle (0.33, p=0.04), Left Ventricular Noncompaction, and other CHD. Shroom3 mutant mice left ventricular wall thickness was 36% thinner compared to wild type mice (99.0±8.6µm, 63.0±8.4µm, p=0.005). LacZ shows the expression of Shroom3 through important stages of cardiac development, and IHC shows actomyosin disruption. In silico analysis demonstrates CHD patients have SHROOM3 variants in highly conserved nucleic acid and protein sequences, and significant protein structural changes.   Conclusion and Potential Impact: Shroom3 null mice have cardiac defects resembling a Wnt/PCP disruption phenotype. Similarly, patients with CHD have likely pathogenic variants in SHROOM3. These data support a role for SHROOM3 in CHD pathogenesis and begin to elucidate mechanisms. Identifying SHROOM3’s role in CHD is critical to understanding cardiac development as well as the diagnosis, management and treatment of CHD.