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 calciumsensitive 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.CNS trauma | live imaging | calcium influx | autophagy A xonal degeneration plays a pivotal role in the pathogenesis of numerous neurological disorders frequently preceding neuronal cell death and resulting in persistent functional disability. Traumatic spinal cord or peripheral nerve injury represent classical conditions where mechanical disruption of axonal integrity results in nervous system dysfunction (1, 2). Several degenerative CNS diseases show prominent axonal pathology already early in the disease course, such as the degeneration of nigrostriatal projection tracts or cardiac sympathetic nerves in Parkinson's disease (3) or corticospinal tracts in amyotrophic lateral sclerosis (4). Key features of axonal degeneration seem to be similar despite variable etiology. The distal part of the lesioned axon undergoes Wallerian degeneration (WD) characterized by initial axonal stability followed by rapid degeneration, fragmentation, and blebbing of the remaining axon, microtubule disassembly, and phagocytic clearance of the lesion site. The proximal part was reported to remain more stable than its distal counterpart (5-8), but imaging of the spinal cord in vivo visualized mechanisms of acute axonal degeneration (AAD) within the first minutes after lesion. In contrast to WD, AAD results in sudden axonal disintegration and extended for ≈300 μm proximal and distal to the lesion (9). One of the putative initiating steps in axonal degeneration is the influx of extracellular calcium, which is suggested to destabilize the axon and to transmit apoptotic signals to the neuronal soma (10-12).The optic nerve (ON) represents a unique model system for the study of axonal pathology in the CNS because of its accessibility and the possibility to manipulate the system...

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.The obligate intracellular parasite Toxoplasma gondii infects virtually any warm-blooded vertebrate and ϳ25% of the world's human population (27). Most infections generate few or no symptoms. Yet, acute infections are a concern in human medicine, since this opportunistic pathogen causes severe neurological complications in immunocompromised individuals, disseminated congenital infections in the developing fetus, and ocular manifestations in otherwise healthy individuals (27). After ingestion of the parasite, acute infection is characterized by the proliferation of fast-growing stages (tachyzoites) that rapidly disseminate and differentiate into slow-growing stages (bradyzoites) in peripheral tissues, where they may persist for the lifetime of the host (27). In contrast to pathogens that rely on uptake by host cells, T. gondii actively invades host cells, including cells of the immune system, and replicates in a nonfusigenic parasitophorous vacuole (45).Mounting evidence indicates that dendritic cells (DC) play critical roles during T. gondii infection as early sources of protective interleukin-12 responses and mediators of antigen presentation (32,35,37,41). In addition, based on their migratory properties (40) and permissiveness to Toxoplasma infection (6), DC have recently been identified as systemic carriers (Trojan horses) of T. gondii tachyzoites (2,7,30). Yet, the precise roles of ...

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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.