No abstract

Granzymes are a family of serine proteases that are harbored in cytoplasmic granules of activated T lymphocytes and are released upon target cell interaction. Immediate and complete neurite retraction was induced in a mouse neuronal cell line when total extracts of granule proteins were added. This activity was isolated and identified as granzyme A. This protease not only induced neurite retraction at nanomolar concentrations but also reversed the stellation of astrocytes. Both effects were critically dependent on the esterolytic activity of granzyme A. As neurite retraction is known to be induced by thrombin, possible cleavage and activation of the thrombin receptor were investigated. A synthetic peptide spanning the N-terminal thrombin receptor activation sequence was cleaved by granzyme A at the authentic thrombin cleavage site Leu-Asp-Pro-Arg -Ser. Antibodies to the thrombin receptor inhibited both thrombin and granzyme A-mediated neurite retraction. Thus, T-cell-released granzyme A induces cellular responses by activation of the thrombin receptor. As braininfiltrating CD4+ lymphocytes are the effector cells in experimental allergic encephalomyelitis, granzyme A released in the brain may contribute to the etiology of autoimmune disorders in the nervous system.

HDs racI Thrombin causes profound metabolic and morphological changes in cultured neural cells via activation of the thrombin receptor, also called protease-activated receptor 1 (PAR1). PAR1 mRNA is present in the rat brain, but the role of this receptor in the nervous system remains elusive. The expression of PAR1 and the potent thrombin inhibitor protease nexin-1 (PN-1) was investigated in the developing rat brain and spinal cord and after peripheral nerve lesion. As seen by in situ hybridization, the PAR1 mRNA signal in the late embryonic and early postnatal nervous system was widespread, but generally of low intensity whereas in the adult it was more pronounced and confined to particular neuronal cells. These include the mesencephalic dopaminergic neurons, several thalamic and brainstem nuclei, the mitral cells in the olfactory bulb and the Purkinje cells in the cerebellum. In the spinal cord, PAR1 mRNA was abundant in motoneurons and a particularly high expression was detected in the preganglionic neurons of the autonomic nervous system. High PAR1 mRNA expression was also found in the dorsal root ganglia. Interestingly, strong immunoreactivity for the protease inhibitor PN-1 was present in spinal motoneuron cell bodies, although its transcript was undetectable there. In response to sciatic nerve transection, the signal intensity of PAR1 mRNA as seen by Northern analysis increased in the proximal and the distal part of the lesioned nerve and in the denervated muscle, whereas the PN-1 mRNA signal strongly increased only in the distal part of the nerve but remained unchanged in the proximal part and in the muscle. After facial nerve transection, PAR1 mRNA expression substantially decreased in facial motoneurons. No PAR1 transcript was detected in reactive astrocytes. Similar to PAR1, PN-1 mRNA which was expressed in interneurons within the facial nucleus was also decreased following facial nerve transection.

Astrocytes are typically star shaped cells playing diverse roles in the function of the nervous system. In astrocyte cultures established from the cerebral hemispheres of newborn rats, the cells have generally a polygonal fibroblast‐like morphology, but acquire a stellate shape upon serum removal. When the serine protease thrombin or the bioactive lipid lysophosphatidic acid is added, the stellate cells revert to the flat morphology. Here we show that the effect of these agents is mediated via activation of the small GTP‐binding protein Rho. Neither thrombin nor lysophosphatidic acid induced spreading of astrocytes microinjected with C3 transferase, an exoenzyme which ADP‐ribosylates and thereby inactivates Rho. In contrast, the response of cells injected with a dominant negative form of Rac was unaffected. In addition, the injection of active Rho into stellate astrocytes mimicked the effect of thrombin and lysophosphatidic acid and an injection of C3 into flat cells grown in serum induced stellation. The conversion from a stellate to a spread morphology upon activation of Rho resulted in the formation of stress fibers and focal adhesions which most probably are key events in establishing and stabilizing the altered cytoarchitecture. These results suggest that Rho plays a crucial role in determining the shape of astrocytes and thereby may modulate their interaction with neurons in vivo. GLIA 21:244–252, 1997. © 1997 Wiley‐Liss, Inc.