DNMT1 regulates expression of MHC class I in post-mitotic neurons

Abstract Major Histocompability Complex I (MHC-I) molecules present cellularly derived peptides to the adaptive immune system. Generally MHC-I is not expressed on healthy post-mitotic neurons in the central nervous system, but it is known to increase upon immune activation such as viral infections and also during neurodegenerative processes. MHC-I expression is known to be regulated by the DNA methyltransferase DNMT1 in non-neuronal cells. Interestingly DNMT1 expression is high in neurons despite these being non-dividing. This suggests a role for DNMT1 in neurons beyond the classical re-methylation of DNA after cell division. We thus investigated whether DNMT1 regulates MHC-I in post-mitotic neurons. For this we used primary cultures of mouse cerebellar granule neurons (CGNs). Our results showed that knockdown of DNMT1 in CGNs caused upregulation of some, but not all subtypes of MHC-I genes. This effect was synergistically enhanced by subsequent IFNγ treatment. Overall MHC-I protein level was not affected by knockdown of DNMT1 in CGNs. Instead our results show that the relative MHC-I expression levels among the different MHC subtypes is regulated by DNMT1 activity. In conclusion, we show that while the mouse H2-D1/L alleles are suppressed in neurons by DNMT1 activity under normal circumstances, the H2-K1 allele is not. This finding is particularly important in two instances. One: in the context of CNS autoimmunity with epitope presentation by specific MHC-I subtypes where this allele specific regulation might become important; and two: in amyotropic lateral sclerosis (ALS) where H2-K but not H2-D protects motor neurons from ALS astrocyte-induced toxicity in a mouse model of ALS

Genetic ablation of soluble TNF does not affect lesion size and functional recovery after moderate spinal cord injury in mice

Traumatic spinal cord injury (SCI) is followed by an instant increase in expression of the microglial-derived proinflammatory cytokine tumor necrosis factor (TNF) within the lesioned cord. TNF exists both as membrane-anchored TNF (mTNF) and as cleaved soluble TNF (solTNF). We previously demonstrated that epidural administration of a dominant-negative inhibitor of solTNF, XPro1595, to the contused spinal cord resulted in changes in Iba1 protein expression in microglia/macrophages, decreased lesion volume, and improved locomotor function. Here, we extend our studies using mice expressing mTNF, but no solTNF (mTNFΔ/Δ), to study the effect of genetic ablation of solTNF on SCI. We demonstrate that TNF levels were significantly decreased within the lesioned spinal cord 3 days after SCI in mTNFΔ/Δ mice compared to littermates. This decrease did, however, not translate into significant changes in other pro- and anti-inflammatory cytokines (IL-10, IL-1β, IL-6, IL-5, IL-2, CXCL1, CCL2, or CCL5), despite a tendency towards increased IL-10 and decreased IL-1β, TNFR1, and TNFR2 levels in mTNFΔ/Δ mice. In addition, microglial and leukocyte infiltration, activation state (Iba1, CD11b, CD11c, CD45, and MHCII), lesion size, and functional outcome after moderate SCI were comparable between genotypes. Collectively, our data demonstrate that genetic ablation of solTNF does not significantly modulate postlesion outcome after SCI

A novel multicolor flow-cytometry application for quantitative detection of receptors on vascular smooth muscle cells

There is a need to develop new techniques for quantitative measurement of receptors expression on particular vasculature cells types. Here, we describe and demonstrate a novel method to measure quantitatively and simultaneously the expression of endothelin B receptor (ETB) on vascular smooth muscle cells (VSMC). We isolated cells from male rat tissues such as: brain pial, brain intraparenchymal and retina vessels. To analyze solid tissues, a single-cell suspension was prepared by a combined mechanic and enzymatic process. The cells were stained with Fixable Viability Dye, followed by fixation, permeabilization and antibodies staining. The expression of ETB receptors on VSMC was measured by flow-cytometry and visualized by fluorescence microscopy. We obtained a high percentage of viable cells 87.6% ± 1.5% pial; 84.6% ± 4.3% parenchymal and 90.6% ± 4% retina after isolation of single cells. We performed a quantitative measurement of ETB receptor expression on VSMC and we identified two subpopulations of VSMC based on their expression of smooth muscle cells marker SM22α. The results obtained from pial vessels are statistically significant (38.4% ± 4% vs 9.8% ± 3.32%) between the two subpopulations of VSMC. The results obtained from intraparenchymal and retina vessels were not statistically significant. By specific gating on two subpopulations, we were able to quantify the expression of ETB receptors. The two subpopulation expressed the same level of ETB receptor (p = 0.45; p = 0.3; p = 0.42) in pial, parenchymal and retina vessels, respectively. We applied our method to the animals after induction of subarachnoid hemorrhage (SAH). There was statistically significant expression of ETB receptor (p = 0.02) on VSMC between sham 61.4% ± 4% and SAH 77.4% ± 4% rats pial vessels. The presented technique is able to quantitatively and selectively measure the level of protein expression on VSMC. The entire technique is optimized for rat tissue; however the protocol can also be adapted for other species