Mechanisms of acute axonal degeneration in the optic nerve in vivo.

Axonal degeneration is an initial key step in traumatic and neurodegenerative CNS disorders. We established a unique in vivo epifluorescence imaging paradigm to characterize very early events in axonal degeneration in the rat optic nerve. Single retinal ganglion cell axons were visualized by AAV-mediated expression of dsRed and this allowed the quantification of postlesional acute axonal degeneration (AAD). EM analysis revealed severe structural alterations of the cytoskeleton, cytoplasmatic vacuolization, and the appearance of autophagosomes within the first hours after lesion. Inhibition of autophagy resulted in an attenuation of acute axonal degeneration. Furthermore, a rapid increase of intraaxonal calcium levels following crush lesion could be visualized using a calcium-sensitive dye. Application of calcium channel inhibitors prevented crush-induced calcium increase and markedly attenuated axonal degeneration, whereas application of a calcium ionophore aggravated the degenerative phenotype. We finally demonstrate that increased postlesional autophagy is calcium dependent and thus mechanistically link autophagy and intraaxonal calcium levels. Both processes are proposed to be major targets for the manipulation of axonal degeneration in future therapeutic settings.

The Toxoplasma gondii-shuttling function of dendritic cells is linked to the parasite genotype.

Following intestinal invasion, the processes leading to systemic dissemination of the obligate intracellular protozoan Toxoplasma gondii remain poorly understood. Recently, tachyzoites representative of type I, II and III T. gondii populations were shown to differ with respect to their ability to transmigrate across cellular barriers. In this process of active parasite motility, type I strains exhibit a migratory capacity superior to those of the type II and type III strains. Data also suggest that tachyzoites rely on migrating dendritic cells (DC) as shuttling leukocytes to disseminate in tissue, e.g., the brain, where cysts develop. In this study, T. gondii tachyzoites sampled from the three populations were allowed to infect primary human blood DC, murine intestinal DC, or in vitro-derived DC and were compared for different phenotypic traits. All three archetypical lineages of T. gondii induced a hypermigratory phenotype in DC shortly after infection in vitro. Type II (and III) strains induced higher migratory frequency and intensity in DC than type I strains did. Additionally, adoptive transfer of infected DC favored the dissemination of type II and type III parasites over that of type I parasites in syngeneic mice. Type II parasites exhibited stronger intracellular association with both CD11c(+) DC and other leukocytes in vivo than did type I parasites. Altogether, these findings suggest that infected DC contribute to parasite propagation in a strain type-specific manner and that the parasite genotype (type II) most frequently associated with toxoplasmosis in humans efficiently exploits DC migration for parasite dissemination.

Transmission of Toxoplasma gondii from infected dendritic cells to natural killer cells.

The obligate intracellular parasite Toxoplasma gondii can actively infect any nucleated cell type, including cells from the immune system. In the present study, we observed that a large number of natural killer (NK) cells were infected by T. gondii early after intraperitoneal inoculation of parasites into C57BL/6 mice. Interestingly, one mechanism of NK cell infection involved NK cell-mediated targeting of infected dendritic cells (DC). Perforin-dependent killing of infected DC led to active egress of infectious parasites that rapidly infected adjacent effector NK cells. Infected NK cells were not efficiently targeted by other NK cells. These results suggest that rapid transfer of T. gondii from infected DC to effector NK cells may contribute to the parasite's sequestration and shielding from immune recognition shortly after infection.

Toxoplasma gondii inhibits Fas/CD95-triggered cell death by inducing aberrant processing and degradation of caspase 8.

Ligation of the death receptor Fas/CD95 activates an apoptotic cascade and plays critical roles during infectious diseases. Previous work has established that infection with the intracellular parasite Toxoplasma gondii renders cells resistant to multiple inducers of apoptosis. However, the effect of T. gondii on the death receptor pathway is poorly characterized. Here we have determined the impact of the parasite on apoptosis in type I cells that transduce Fas/CD95 engagement via the death receptor pathway without the need of a mitochondrial amplification loop. The results have shown that T. gondii significantly reduced Fas/CD95-triggered apoptosis by impairing activation of the initiator caspase 8. Parasitic infection diminished the cellular amount of procaspase 8, resulting in its decreased recruitment to the death-inducing signalling complex and the impaired activation of effector caspases. Remarkably, downregulation of caspase 8 protein in T. gondii-infected cells also occurred in the absence of Fas/CD95 engagement and was associated with the appearance of non-canonical caspase 8 cleavage fragments. Distinct parasite proteins were associated with caspase 8 and its proteolytic fragments. These findings indicate that T. gondii aberrantly processes and finally degrades the initiator caspase 8, thereby, blocking Fas/CD95-mediated apoptosis which signals independently of the apoptogenic function of host cell mitochondria.