Changes in parvalbumin immunoreactive retinal ganglion cells and amacrine cells after optic nerve injury.

Parvalbumin (PARV) is a Ca(2+)-binding protein that may offer resistance to cell death as it primarily functions to maintain Ca(2+) homeostasis. The purpose of this study was to investigate whether PARV expressing retinal ganglion cells (RGCs) would be more resistant to cell death than RGCs that do not express PARV. RGCs in Sprague-Dawley rats were retrogradely labeled with Fluorogold (FG). After 2-28 days following an optic nerve crush (ONC) injury immunohistochemistry was performed on the sections using antibodies against PARV and markers of RGCs. The proportion of retinal ganglion cell layer cells labeled with PARV colocalized with FG or Brn3a and labeled only with PARV (displaced amacrine cells; dACs) were analyzed. PARV staining intensity was measured in ACs, dACs, and RGCs. Double labeling studies revealed that 49% of RGCs and 22% of dACs expressed PARV. There was an immediate reduction in RGC PARV staining after ONC but the overall rate of cell death after 28 days was similar in PARV and non-PARV expressing RGCs. There was no change in PARV AC or dAC number or staining intensity. Although this study suggests that there is no selective survival of the subpopulation of RGCs that contain PARV, there is down-regulation of PARV expression by these RGCs. This suggests that down-regulation of PARV may contribute to RGC death due to a compromised Ca(2+) buffering capacity. Maintaining PARV expression after injury could be an important neuroprotective strategy to improve RGC survival after injury.

Identification of the Mycobacterium tuberculosis protein PE-PGRS62 as a novel effector that functions to block phagosome maturation and inhibit iNOS expression.

Using a genetic screen in yeast we found that Mycobacterium tuberculosis PE-PGRS62 was capable of disrupting yeast vacuolar protein sorting, suggesting effects on endosomal trafficking. To study the impact of PE-PGRS62 on macrophage function, we infected murine macrophages with Mycobacterium smegmatis expressing PE-PGRS62. Infected cells displayed phagosome maturation arrest. Phagosomes acquired Rab5, but displayed a significant defect in Rab7 and LAMP-1 acquisition. Macrophages infected with M. smegmatis expressing PE-PGRS62 also expressed two- to threefold less iNOS protein when compared with cells infected with wild-type bacteria. Consistent with this, cells infected with a Mycobacterium marinum transposon mutant for the PE-PGRS62 orthologue showed greater iNOS protein expression when compared to cells infected with wild-type organisms. Complementation restored the ability of the mutant to inhibit iNOS expression. No differences in iNOS transcript levels were observed, suggesting that PE-PGRS62 effects on iNOS expression occurred post-transcriptionally. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum transposon mutant, suggesting that PE-PGRS62 may affect cell wall composition. These findings suggest that PE-PGRS62 supports virulence via inhibition of phagosome maturation and iNOS expression, and these phenotypes may be linked to effects on bacterial cell wall composition.