SMPD4 is a neutral sphingomyelinase implicated in a specific type of congenital microcephaly. Although not intensively studied, SMPD4 deficiency has also been found to cause cell division defects. This suggests a role for SMPD4 in cell-cycle and differentiation. In order to explore this role, we used proximity ligation to identify the partners of SMPD4 in vivo in HEK293T cells. We found that these partners localize near the endoplasmic reticulum (ER) and the nuclear membrane. Using mass spectrometry, we could identify these partners and discovered that SMPD4 is closely associated with several nucleoporins, including NUP35, a nucleoporin directly involved in pore membrane curvature and pore insertion. This suggests that SMPD4 may play a role in this process.

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation, dendritic and axonal growth. The relevance of ER-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by biallelic loss of function variants in Thioredoxin (TRX)Related Transmembrane-2 ( TMX2 ), detected by exome sequencing in fourteen affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria and other migration disorders. TMX2 encodes one of the five TMX proteins of the Protein Disulfide Isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER Mitochondria-Associated-Membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2 -deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration and organization in the developing brain. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Sophia Children's Hospital Department of Clinical Genetics Postal address P.O. box 2060 3000 CB Rotterdam, NL Visiting address Dr. Molewaterplein 40 3015 GD Rotterdam, NL Contact & route www.erasmusmc.nl Head of Department Prof. dr. R.M.W. Hofstra, PhD Heads of section Mr R.J.H. Galjaard, MD, PhD www.erasmusmc.nl To The Editor of the American Journal of Human Genetics Dr Sara Cullinan Direct dial +31-10-7036915 Fax number +31-10-7043072 Internal postal address Ee2018 E-mail ervo@erasmusmc.nl Our reference AJHG-D-19-00366-R4 Date October 3, 2019 Concerning AJHG-D-19-00366R3 , manuscript title: TMX2 is a key regulator of cellular redox state and its dysfunction causes severe brain developmental abnormalities.

The redox state of the neural progenitors regulates physiological processes such as neuronal differentiation and dendritic and axonal growth. The relevance of endoplasmic reticulum (ER)-associated oxidoreductases in these processes is largely unexplored. We describe a severe neurological disorder caused by bi-allelic loss-of-function variants in thioredoxin (TRX)-related transmembrane-2 (TMX2); these variants were detected by exome sequencing in 14 affected individuals from ten unrelated families presenting with congenital microcephaly, cortical polymicrogyria, and other migration disorders. TMX2 encodes one of the five TMX proteins of the protein disulfide isomerase family, hitherto not linked to human developmental brain disease. Our mechanistic studies on protein function show that TMX2 localizes to the ER mitochondria-associated membranes (MAMs), is involved in posttranslational modification and protein folding, and undergoes physical interaction with the MAM-associated and ER folding chaperone calnexin and ER calcium pump SERCA2. These interactions are functionally relevant because TMX2-deficient fibroblasts show decreased mitochondrial respiratory reserve capacity and compensatory increased glycolytic activity. Intriguingly, under basal conditions TMX2 occurs in both reduced and oxidized monomeric form, while it forms a stable dimer under treatment with hydrogen peroxide, recently recognized as a signaling molecule in neural morphogenesis and axonal pathfinding. Exogenous expression of the pathogenic TMX2 variants or of variants with an in vitro mutagenized TRX domain induces a constitutive TMX2 polymerization, mimicking an increased oxidative state. Altogether these data uncover TMX2 as a sensor in the MAM-regulated redox signaling pathway and identify it as a key adaptive regulator of neuronal proliferation, migration, and organization in the developing brain.