Granule-derived granzyme B mediates the vulnerability of human neurons to T cell-induced neurotoxicity.

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.

Trafficking of TRPP2 by PACS proteins represents a novel mechanism of ion channel regulation.

The trafficking of ion channels to the plasma membrane is tightly controlled to ensure the proper regulation of intracellular ion homeostasis and signal transduction. Mutations of polycystin-2, a member of the TRP family of cation channels, cause autosomal dominant polycystic kidney disease, a disorder characterized by renal cysts and progressive renal failure. Polycystin-2 functions as a calcium-permeable nonselective cation channel; however, it is disputed whether polycystin-2 resides and acts at the plasma membrane or endoplasmic reticulum (ER). We show that the subcellular localization and function of polycystin-2 are directed by phosphofurin acidic cluster sorting protein (PACS)-1 and PACS-2, two adaptor proteins that recognize an acidic cluster in the carboxy-terminal domain of polycystin-2. Binding to these adaptor proteins is regulated by the phosphorylation of polycystin-2 by the protein kinase casein kinase 2, required for the routing of polycystin-2 between ER, Golgi and plasma membrane compartments. Our paradigm that polycystin-2 is sorted to and active at both ER and plasma membrane reconciles the previously incongruent views of its localization and function. Furthermore, PACS proteins may represent a novel molecular mechanism for ion channel trafficking, directing acidic cluster-containing ion channels to distinct subcellular compartments.

Chromatin domain boundaries delimited by a histone-binding protein in yeast.

When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.