Abstract
Increasing evidence supports a critical role of T cells in neurodegeneration associated with acute and subacute brain inflammatory disorders. Granzyme B (GrB), released by activated T cells, is a cytotoxic proteinase which may induce perforin-independent neurotoxicity. Here, we studied the mechanism of perforin-independent GrB toxicity by treating primary cultured human neuronal cells with recombinant GrB. GrBactivated the protease-activated receptor (PAR)-1 receptor on the neuronal cell surface leading to decreased intracellular cyclic AMP levels. This was followed by increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane. Similar expression of Kv1.3 was also seen in neurons of the cerebral cortex adjacent to active inflammatory lesions in patients with multiple sclerosis. Kv1.3 expression was followed by activation of Notch-1 resulting in neurotoxicity. Blocking PAR-1, Kv1.3 or Notch-1 activation using specific pharmacological inhibitors or siRNAs prevented GrB-induced neurotoxicity. Furthermore, clofazimine protected against GrB-induced neurotoxicity in rat hippocampus, in vivo. These observations indicate that GrB released from T cells induced neurotoxicity by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1. These pathways provide novel targets to treat T cell-mediated neuroinflammatory disorders. Kv1.3 is of particular interest since it is expressed on the cell surface, only under pathological circumstances, and early in the cascade of events making it an attractive therapeutic target.
Highlights
Tcell activation plays a critical role in brain inflammatory mechanisms and related neurodegeneration such as brain ischemic/reperfusion injury [1], multiple sclerosis (MS), acute disseminated encephalomyelitis, Rasmussen encephalitis [2], and HIV 1-associated immune reconstitution syndrome [3]
We observed that using a chamber culturing system which separated neuronal cell bodies and axons, the supernatants from activated T cells caused more significant damage to axons when they were added to the cell body side than when they were applied to the axon side (Figure S1)
We used primary cultures of human neuronal cells to investigate the mechanisms of granzyme B (GrB)-induced neurotoxicity
Summary
Tcell activation plays a critical role in brain inflammatory mechanisms and related neurodegeneration such as brain ischemic/reperfusion injury [1], multiple sclerosis (MS), acute disseminated encephalomyelitis, Rasmussen encephalitis [2], and HIV 1-associated immune reconstitution syndrome [3]. Activated T cells induce neurotoxicity through both cell contact-dependent [5] and –independent pathways such as release of granzyme B (GrB) [6]. GrB may cause perforin-independent cytotoxicity by using membrane receptors such as mannose-6phosphate receptor and heparan sulfate containing receptor to facilitate its entry into the cells [7,8] or by cleaving or interacting with the membrane-bound receptors such as glutamate receptor GluR3 [9] and Notch-1 [10] without entry into the cell. Notch-1 activation in neurons is related to neurotoxicity, especially neurite damage [11,12]. We found that GrB-mediated neurotoxicity is pertussis toxin (PTX)-dependent [6], indicating a novel mechanism involving a G protein-coupled receptor (GPCR)
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