The cause of cortical neurodegeneration and subsequent grey matter (GM) atrophy is not completely understood, although it is suggested to be stimulated by a cascade of events triggered by chronic inflammation. Transcriptomics analysis of cortical GM suggests a dysregulation of TNF signaling towards activation of necroptosis in the presence of increased meningeal inflammation.
To explore the role of sustained production of TNF in the subarachnoid space/meninges as a trigger for the activation of TNFR1-dependent necroptosis and subsequent neurodegeneration in progressive MS.
Quantitation of levels of proteins involved in necroptotic signaling was carried out in post-mortem brains of MS cases (n=28) and controls (n=11) by western blotting, together with localisation by immunohistochemistry. A rat model of cortical pathology in which TNF and IFNg are chronically over-expressed in the cortical meninges was used to determine the consequences of elevated CSF cytokines on cortical neurons. Primary rat cortical neurons were used of in vitro validation studies.
A substantial significant increase in protein levels for TNFR1 and the subsequent key steps of necroptotic signaling, namely the RIPK1, RIPK3 and MLKL kinase cascade, was present in the MS GM, accompanied by a decrease in the caspase 8 cleaved p18 subunit. Upregulation of these proteins occurred predominantly in pyramidal neurons in cortical layers II-III and V-VI, together with an increase in the percentage of positive neurons, with negligible expression in the control brain. When MS cases were classified according to the severity of meningeal inflammation, pMLKL and MLKL levels were both significantly increased in cases with more abundant meningeal inflammation (6.5 and 8.0-fold respectively). Oligomeric forms of MLKL, which indicate the final stages of necroptosis, were found exclusively in MS GM. Chronic overexpression of TNF and IFNg in the rat cortical meninges gave rise to neuronal loss in cortical layers II-III and a >20-fold increase in neurons expressing necroptotic markers. Finally, exposure of rat primary cultured cortical neurons to TNF induced necroptosis, but only when apoptosis was inhibited.
Taken together, our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated by chronic meningeal inflammation, which contributes to the accumulation of clinical deficit in MS.