Catherine Doller

Astrocytes Regulate Microglial Phagocytosis of Senile Plaque Cores of Alzheimer's Disease ☆

We have developed an in vitro model in which isolated senile plaque (SP) cores are presented to rat microglial cells in culture. Microglia rapidly phagocytosed, broke apart, and cleared SP cores. However, when cocultured with astrocytes, microglial phagocytosis was markedly suppressed, allowing the SPs to persist. Suppression of phagocytosis by astrocytes appears to be a general phenomena since microglia in the presence of astrocytes showed reduced capacity to phagocytose latex beads as well. The astrocyte effect on microglia is related in part to a diffusible factor(s) since astrocyte- but not fibroblast-conditioned media also reduced phagocytosis. These results suggest that while microglia have the capacity to phagocytose and remove SPs, astrocytes which lie in close association to microglia may help prevent the efficient clearance of SP material allowing them to persist in Alzheimers disease.

Studies on the Development and Behavior of the Dystrophic Growth Cone, the Hallmark of Regeneration Failure, in an In Vitro Model of the Glial Scar and after Spinal Cord Injury

We have developed a novel in vitro model of the glial scar that mimics the gradient of proteoglycan found in vivo after spinal cord injury. In this model, regenerated axons from adult sensory neurons that extended deeply into the gradient developed bulbous, vacuolated endings that looked remarkably similar to dystrophic endings formed in vivo. We demonstrate that despite their highly abnormal appearance and stalled forward progress, dystrophic endings are extremely dynamic both in vitro and in vivo after spinal cord injury. Time-lapse movies demonstrated that dystrophic endings continually send out membrane veils and endocytose large membrane vesicles at the leading edge, which were then retrogradely transported to the rear of the “growth cone.” This direction of movement is contrary to membrane dynamics that occur during normal neurite outgrowth. As further evidence of this motility, dystrophic endings endocytosed large amounts of dextran both in vitro and in vivo. We now have an in vitro model of the glial scar that may serve as a potent tool for developing and screening potential treatments to help promote regeneration past the lesion in vivo.

Astrocyte-Associated Fibronectin Is Critical for Axonal Regeneration in Adult White Matter

Although it has been suggested that astroglia guide pioneering axons during development, the cellular and molecular substrates that direct axon regeneration in adult white matter have not been elucidated. We show that although adult cortical neurons were only able to elaborate very short, highly branched, dendritic-like processes when seeded onto organotypic slice cultures of postnatal day 35 (P35) rat brain containing the corpus callosum, adult dorsal root ganglion (DRG) neurons were able to regenerate lengthy axons within the reactive glial environment of this degenerating white matter tract. The callosum in both P35 slices and adult rat brain was rich in fibronectin, but not laminin. Furthermore, the fibronectin was intimately associated with the intratract astrocytes. Blockade of fibronectin function in situ with an anti-fibronectin antibody dramatically decreased outgrowth of DRG neurites, suggesting that fibronectin plays an important role in axon regeneration in mature white matter. The critical interaction between regrowing axons and astroglial-associated fibronectin in white matter may be an additional factor to consider when trying to understand regeneration failure and devising strategies to promote regeneration.

The Critical Role of Basement Membrane-Independent Laminin γ1 Chain during Axon Regeneration in the CNS

We have addressed the question of whether a family of axon growth-promoting molecules known as the laminins may play a role during axon regeneration in the CNS. A narrow sickle-shaped region containing a basal lamina-independent form of laminin exists in and around the cell bodies and proximal portion of the apical dendrites of CA3 pyramidal neurons of the postnatal hippocampus. To understand the possible function of laminin in axon regeneration within this pathway, we have manipulated laminin synthesis at the mRNA level in a slice culture model of the lesioned mossy system. In this model early postnatal mossy fibers severed near the hilus can regenerate across the lesion and elongate rapidly within strata lucidum and pyramidale. In slice cultures of the postnatal day 4 hippocampus, 2 d before lesion and then continuing for 1–5 d after lesion, translation of the γ1 chain product of laminin was reduced by using antisense oligodeoxyribonucleotides and DNA enzymes. In the setting of the lesioned organotypic hippocampal slice, astroglial repair of the lesion and overall glial patterning were unperturbed by the antisense or DNA enzyme treatments. However, unlike controls, in the treated, lesioned slices the vast majority of regenerating mossy fibers could not cross the lesion site; those that did were very much shorter than usual, and they took a meandering course. In a recovery experiment in which the DNA enzyme or antisense oligos were washed away, laminin immunoreactivity returned and mossy fiber regeneration resumed. These results demonstrate the critical role of laminin(s) in an axon regeneration model of the CNS.

Myeloid Suppressor Cells Induced by Retinal Pigment Epithelial Cells Inhibit Autoreactive T-Cell Responses That Lead to Experimental Autoimmune Uveitis

PURPOSE To test whether retinal pigment epithelial (RPE) cells are able to induce myeloid-derived suppressor cell (MDSC) differentiation from bone marrow (BM) progenitors. METHODS BM cells were cocultured with or without RPE cells in the presence of GM-CSF and IL-4. Numbers of resultant MDSCs were assessed by flow cytometry after 6 days of incubation. The ability of the RPE cell-induced MDSCs to inhibit T cells was evaluated by a CFSE-based T-cell proliferation assay. To explore the mechanism by which RPE cells induce MDSC differentiation, PD-L1-deficient RPE cells and blocking antibodies against TGF-β, CTLA-2α, and IL-6 were used. RPE cell-induced MDSCs were adoptively transferred into mice immunized with interphotoreceptor retinoid-binding protein in complete Freunds adjuvant to test their efficacy in suppressing autoreactive T-cell responses in experimental autoimmune uveitis (EAU). RESULTS RPE cells induced the differentiation of MDSCs. These RPE cell-induced MDSCs significantly inhibited T-cell proliferation in a dose-dependent manner. PD-L1-deficient RPE cells induced MDSC differentiation as efficiently as wild-type RPE cells, and neutralizing TGF-β or CTLA-2α did not alter the numbers of induced MDSCs. However, blocking IL-6 reduced the efficacy of RPE cell-induced MDSC differentiation. Finally, adoptive transfer of RPE cell-induced MDSCs suppressed IRBP-specific T-cell responses that led to EAU. CONCLUSIONS RPE cells induce the differentiation of MDSCs from bone marrow progenitors. Both cell surface molecules and soluble factors are important in inducing MDSC differentiation. PD-L1, TGF-β, and CTLA-2α were not measurably involved in RPE cell-induced MDSC differentiation, whereas IL-6 was important in the process. The induction of MDSCs could be another mechanism by which RPE cells control immune reactions in the retina, and RPE cell-induced MDSCs should be further investigated as a potential approach to therapy for autoimmune posterior uveitis.

Indoleamine 2,3-dioxygenase overexpression causes kynurenine-modification of proteins, fiber cell apoptosis and cataract formation in the mouse lens

Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the kynurenine pathway. The kynurenines formed in this pathway chemically modify proteins and cause apoptosis in cells. Evidence suggests that kynurenines and their protein modifications are involved in cataract formation, but this has yet to be directly demonstrated. We generated transgenic (Tg) mouse lines that overexpress human IDO in the lens. Homozygous Tg (homTg) lenses had higher IDO immunoreactivity, ∼4.5 times greater IDO mRNA, and ∼8 times higher IDO activity compared to lenses from hemizygous Tg (hemTg) animals. The kynurenine content was threefold higher in homTg than in hemTg but was not detected in wild-type (Wt) lenses. Kynurenine modifications were ∼2.6 times greater in homTg than in hemTg or Wt. HomTg lenses had vacuoles in the epithelium and cortical fiber cells. Kynurenine modifications coincided with apoptosis in the secondary fiber cells of homTg lenses. Caspase-3 and caspase-9 activities were markedly higher in homTg than in hemTg and Wt. The glutathione content was ∼36% lower in homTg compared to hemTg and Wt lenses. HomTg animals also developed bilateral cataracts within 3 months of birth. Together these data demonstrate that IDO-mediated production of kynurenines results in defects in fiber cell differentiation and their apoptosis and suggest that IDO activity is kept low in the lens to prevent deleterious effects by kynurenines.