The most striking distinction was the increased expression of TREX1 in the white matter of the cerebral cortex from brain tissue of RVCL cases (5.5 1.9% of Iba1+ microglia in normal controls vs. intron in transcript variant 1. C. Same as B with primers designed to detect transcript variant 1 only as a ~300 base pair product. BPA-28-806-s005.tif (12M) GUID:?4A25646B-A5CC-462E-A046-8DF5066654B5 Figure S2. TREX1 expressing cells are not astrocytes. Dual staining of formalin\fixed, paraffin\embedded human brain tissue from a case of RVCL with anti\glial fibrillary acidic protein (GFAP, green), a marker for astrocytes, and anti\TREX1 (reddish). Scale bar represents 21 m. BPA-28-806-s004.tiff (783K) GUID:?4E9EE2BB-66AD-428D-A8A7-01A4F2640972 Physique S3. Complete cell counts for Iba1 and TREX1 by brain region in normal controls and cases of RVCL and ischemic stroke. The cell counts for Iba1 (A) and TREX1 (B) for specific areas of the brain in normal controls (blue bars), cases with RVCL (reddish bars) and cases with ischemic stroke (green bars). Sections quantified in RVCL and ischemic stroke were taken from undamaged tissue. The number of tissue sections included for each observation is usually noted above each column. White matter (WM); Gray matter (GM). Data are offered as mean SEM. BPA-28-806-s003.tif (934K) GUID:?3FA95726-146E-40EE-A518-86D30DED4F02 Physique S4. Representative immunohistochemical staining for TREX1 (brown) in normal controls (left panel) and undamaged tissue in RVCL cases (right panel) in specified brain regions. Nuclei are counterstained with hematoxylin (blue). Level bar represents 100 m. BPA-28-806-s006.tiff (1.9M) GUID:?EE739987-D6DA-4BBC-8C6E-8F9EBC3674C8 Figure S5. TREX1 cells are ameboid microglia or infiltrating peripheral macrophages along the edges of ischemic lesions. Dual staining of formalin\fixed, paraffin\embedded human brain tissue from a case of ischemic stroke with RCA\1 (left panel, green), a microglial/macrophage and endothelial Manidipine 2HCl cell marker, and anti\TREX1 (reddish). Nuclei are counterstained with TO\PRO\3 (blue). Level bar represents 28 m. BPA-28-806-s002.tif (1.5M) GUID:?326E5B24-65F6-402C-9807-6A7629688DF6 Table S1. Summary of the tissue sections analyzed. BPA-28-806-s001.docx (71K) GUID:?350515D5-967F-44F3-88EA-CBBA21D1D968 Abstract Background Mutations Manidipine 2HCl in the three\prime repair exonuclease 1 (mutations, AicardiCGouitres syndrome (AGS), systemic lupus erythematosus (SLE), and familial chilblain lupus (FCL) 3, 13, 14, 20. In each of these diseases, mutations usually decrease or abolish exonuclease function. AGS mimics Manidipine 2HCl a congenital viral contamination of the brain and typically manifests as a neurodevelopmental disorder in early infancy. As the extra\neurological findings in AGS overlap with SLE, AGS has been proposed as a genetic model of systemic autoimmunity 14. RVCL though does not clinically or pathologically resemble an autoimmune disease but rather a premature degeneration of the microvasculature 26. Most studies have focused on the exonuclease activity of TREX1, for which the carboxy\terminus is not necessary. Recently, it has been shown that this carboxy\terminus Manidipine 2HCl plays a role in suppressing glycan\induced inflammation, a function that is lost by the frame\shift mutations of RVCL 8. More recently, Manidipine 2HCl mice transporting a common RVCL frame\shift mutation have developed autoantibodies although this is infrequently observed in patients with RVCL 15, 26. However, the mechanism by which this mislocalized but enzymatically active exonuclease Rabbit Polyclonal to Gastrin results in a systemic vasculopathy remains elusive. Although TREX1 transcripts have been identified at the RNA level in all tissues examined, the endogenous expression of the protein has not been studied 17. Given the burden of the neurological disease in RVCL, we evaluated the expression of TREX1 in human brain tissue using a rabbit polyclonal antibody (pAb) we produced. With the use of immunohistochemical and immunofluorescent staining, we sought to identify the specific cell types expressing TREX1 in the brain for insight into the relationship between this exonuclease and the microvasculature. Materials and Methods Antibody production As insufficient amounts of recombinant wild\type TREX1 could be recovered by us (unpublished) or others, 10, 16, 17 recombinant V235fs mutant TREX1 was produced in (detailed in the Supplementary Methods). Antibody purification The rabbit pAb to TREX1 was purified by precipitation with saturated ammonium sulfate for use in WB analyses. For immunofluorescence and immunohistochemical staining, the pAb was further purified by antigen affinity to the recombinant immunogen (V235fs). Details are provided in the Supplementary Strategies. The purified Ab arrangements were electrophoresed on the Tris\glycine gel and stained with Coomassie Blue.