Probes for INS

PURPOSE : Methylenetetrahydrofolate reductase (_MTHFR_) is a critical enzyme in the folate/methionine/homocysteine pathway. Variants in _MTHFR, _notably _677C>T,_ have_ _been associated with glaucoma as well as Alzheimer’s disease and vascular dementia, suggesting an overlapping mechanism in brain and eye. However, mechanisms driving increased risk are not known, hindering the development of new treatments. Approximately 30% of individuals carry at least one copy of _MTHFR677C>T_, causing a 50% decrease in MTHFR enzyme efficiency. Reduced efficiency can lead to high levels of homocysteine in blood, resulting in vascular inflammation and increased risk for vascular damage. We hypothesize that vascular-specific expression of _MTHFR677C>T_ drives damaging effects in the retinal vasculature, priming the environment for additional risk.

METHODS : Retinal, optic nerve head (ONH) and distal optic nerve (ON) tissues from 8 juvenile 10-12 week-old cats (4 males and 4 females) with feline congenital glaucoma (FCG) and 5 age-matched normal control cats (3 males and 2 females) were used. Data for weekly intraocular pressure (IOP) and optic nerve axon counts were available for all subjects. Protein and gene expression in tissue cryosections were examined by immunofluorescence labeling (IF) and RNAscope in situ hybridization (ISH), respectively. Retinal tissue was IF labeled for myeloid cell marker, IBA-1 and flat-mounted. ISH for markers of infiltrating monocytes/macrophages (_CCR2_) and proinflammatory cytokines (_IL1A_, _C1QA_, _TNF_) was performed. Microglia were identified by IF of homeostatic microglial marker, P2RY12. Microscopy images wereanalyzed using Image J, QuPath and Imaris. Two-tailed unpaired t-test or Mann-Whitney test or ANOVA were used for between-group comparisons (p

RESULTS : Short-term lineage tracing data showed that _Lrig1_, _Lgr6_ and _Axin2_ label basal cells in MG ducts and acini. Long-term lineage tracing results showed that clones of labeled cells persist through multiple rounds of ductal and acinar renewal and give rise to differentiated progeny, identifying _Lrig1_+, _Lgr6_+ and _Axin2+_ ductal and acinar basal cells as self-renewing SCs. Forced expression of GLI2ΔN enhanced basal proliferation, caused expansion of _Lrig1_+ SCs, and lead to replacement of lipid-filled meibocytes by proliferative and poorly differentiated acinar cells. Transcriptional profiling of GLI2ΔN-expressing and control MGs revealed that forced GLI2ΔN expression caused greatly increased expression of _Lrig1_ and _Lgr6_ and suppressed expression of meibocyte differentiation genes.

* Alfredo Dueñas Rey Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Víctor López-Soriano Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Zelia Corradi Radboudumc Department of Human Genetics, Nijmegen, Gelderland, Netherlands * Claire-Marie Dhaenens Univ. Lille, Inserm, CHU Lille, U1172 - LilNCog - Lille Neuroscience & Cognition, Lille, France * Manon Bouckaert Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Jasper Verwilt Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium OncoRNALab, Cancer Research Institute Ghent, Ghent, Belgium * Avril M Watson Newcastle University Faculty of Medical Sciences, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom * Majlinda Lako Newcastle University Faculty of Medical Sciences, Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom * Eva D’haene Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Karla Alejandra Ruiz Ceja Telethon Institute of Genetics and Medicine, Napoli, Campania, Italy * Sandro Banfi Telethon Institute of Genetics and Medicine, Napoli, Campania, Italy * Miriam Bauwens Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Frans P Cremers Radboudumc Department of Human Genetics, Nijmegen, Gelderland, Netherlands * Steve Lefever Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Elfride De Baere Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium * Frauke Coppieters Universitair Ziekenhuis Gent Centrum Medische Genetica Gent, Gent, Belgium Department of Biomolecular Medicine, Universiteit Gent Faculteit Geneeskunde en Gezondheidswetenschappen, Gent, Belgium