Granule-Derived Granzyme B Mediates the Vulnerability of Human Neurons to T Cell-Induced Neurotoxicity

Abstract

Multiple sclerosis (MS) is considered an autoimmune disease of the CNS and is characterized by inflammatory cells infiltrating the CNS and inducing demyelination, axonal loss, and neuronal death. Recent evidence strongly suggests that axonal and neuronal degeneration underlie the progression of permanent disability in MS. In this study, we report that human neurons are selectively susceptible to the serine-protease granzyme B (GrB) isolated from cytotoxic T cell granules. In vitro, purified human GrB induced neuronal death to the same extent as the whole activated T cell population. On the contrary, activated T cells isolated from GrB knockout mice failed to induce neuronal injury. We found that following internalization through various parts of neurons, GrB accumulated in the neuronal soma. Within the cell body, GrB diffused out of endosomes possibly through a perforin-independent mechanism and induced subsequent activation of caspases and cleavage of α-tubulin. Inhibition of caspase-3, a well-known substrate for GrB, significantly reduced GrB-mediated neurotoxicity. We demonstrated that treatment of neurons with mannose-6-phosphate prevented GrB entry and inhibited GrB-mediated neuronal death, suggesting mannose-6-phosphate receptor-dependent endocytosis. Together, our data unveil a novel mechanism by which GrB induces selective neuronal injury and suggest potential new targets for the treatment of inflammatory-mediated neurodegeneration in diseases such as MS.

Axons are injured by antigen-specific CD8+ T cells through a MHC class I- and granzyme B-dependent mechanism

“…Axoplasmic GzB activity may also serve as a final axon execution mechanism for the indirect injury of neurons and axons. Bystander axon injury is reported in model systems of myelin antigen-directed and astrocyte-targeted CTLs 45-48 and experiments using gang culture neurons demonstrate that purified GzB and exogenously applied lytic granules containing GzB are capable of destabilizing the axon cytoskeleton 49,50 . Therefore, regardless of the mechanism by which lytic granules encounter the axon, either through direct targeting by axon-specific CTLs, as presented in this work, or by collateral diffusion from glia-targeted immune synapses, the introduction of active GzB into the axoplasm may prove singularly sufficient to injure axons.…”

Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

“…Once infiltrated in the brain, inflammatory factors released from T cells may injure neurons or impair the normal functions of local neural stem cells, resulting in loss of functional neurons and delay of recovery [4,5]. We have previously reported that granzyme B (GrB) released from activated T cells inhibits neurogenesis in adult animals and in cultured human fetal neural stem cells.…”

Rab32 connects ER stress to mitochondrial defects in multiple sclerosis

“…Cultures of human fetal neurons (HFN) were generated from 15–19-week fetal brains (obtained with consent from the University of Alberta Ethics Committee) as described [26]. …”

“…Cultures of human fetal neurons (HFN) were obtained with consent from the University of Alberta Ethics Committee as described [26]. MS cerebral sub-ventricular deep white matter samples were obtained from the NeuroResource Tissue Bank, UCL Institute of Neurology, London, and UK MS Biobank, with next-of-kin informed consent for tissue donation and ethical approval from Central London REC1 (I.D.08/H0718/62) and approval for the study from the Local Research Ethics Committee (I.D.04/Q2102/111).…”