Age-related differences in the dynamics of hippocampal proteasome recovery.

Regulation of proteasome abundance to meet cell needs under stress conditions is critical for maintaining cellular homeostasis. However, the effects of aging on this homeostatic response remain unknown. In this report, we analyzed in young and aged rat hippocampus, the dynamics of proteasome recovery induced by proteasome stress. Proteasome inhibition in young rats leads to an early and coordinate transcriptional and translational up-regulation of both the catalytic subunits of constitutive proteasome and the proteasome maturation protein. By contrast, aged rats up-regulated the inducible catalytic subunits and showed a lower and shorter expression of proteasome maturation protein. This resulted in a faster recovery of proteasome activity in young rats. Importantly, proteasome inhibition highly affected pyramidal cells, leading to the accumulation of ubiquitinated proteins in perinuclear regions of aged, but not young pyramidal neurons. These data strongly suggest that age-dependent differences in proteasome level and composition could contribute to neurodegeneration induced by proteasome dysfunction in normal and pathological aging.

Lipopolysaccharide-induced neuroinflammation leads to the accumulation of ubiquitinated proteins and increases susceptibility to neurodegeneration induced by proteasome inhibition in rat hippocampus.

BACKGROUND: Neuroinflammation and protein accumulation are characteristic hallmarks of both normal aging and age-related neurodegenerative diseases. However, the relationship between these factors in neurodegenerative processes is poorly understood. We have previously shown that proteasome inhibition produced higher neurodegeneration in aged than in young rats, suggesting that other additional age-related events could be involved in neurodegeneration. We evaluated the role of lipopolysaccharide (LPS)-induced neuroinflammation as a potential synergic risk factor for hippocampal neurodegeneration induced by proteasome inhibition. METHODS: Young male Wistar rats were injected with 1 µL of saline or LPS (5 mg/mL) into the hippocampus to evaluate the effect of LPS-induced neuroinflammation on protein homeostasis. The synergic effect of LPS and proteasome inhibition was analyzed in young rats that first received 1 µL of LPS and 24 h later 1 µL (5 mg/mL) of the proteasome inhibitor lactacystin. Animals were sacrificed at different times post-injection and hippocampi isolated and processed for gene expression analysis by real-time polymerase chain reaction; protein expression analysis by western blots; proteasome activity by fluorescence spectroscopy; immunofluorescence analysis by confocal microscopy; and degeneration assay by Fluoro-Jade B staining. RESULTS: LPS injection produced the accumulation of ubiquitinated proteins in hippocampal neurons, increased expression of the E2 ubiquitin-conjugating enzyme UB2L6, decreased proteasome activity and increased immunoproteasome content. However, LPS injection was not sufficient to produce neurodegeneration. The combination of neuroinflammation and proteasome inhibition leads to higher neuronal accumulation of ubiquitinated proteins, predominant expression of pro-apoptotic markers and increased neurodegeneration, when compared with LPS or lactacystin (LT) injection alone. CONCLUSIONS: Our results identify neuroinflammation as a risk factor that increases susceptibility to neurodegeneration induced by proteasome inhibition. These results highlight the modulation of neuroinflammation as a mechanism for neuronal protection that could be relevant in situations where both factors are present, such as aging and neurodegenerative diseases.

Regional difference in inflammatory response to LPS-injection in the brain: role of microglia cell density.

To elucidate whether density of cells could contribute to the extent of microglial activation, we performed in vitro assays using three different densities of N13 microglia stimulated with LPS. Our results showed that induction of pro-inflammatory factors as TNF-α and iNOS was directly related to cell density, meanwhile the induction of the anti-inflammatory IL-10 was inversely related to cell density. Accordingly, in vivo assays showed that after LPS-injection, iNOS expression was more intense in substantia nigra, a brain area showing specific susceptibility to neurodegeneration after microglia activation, whereas IL-10 expression was more sustained in striatum, an area resistant to damage. These results support that microglia density is pivotal to control the balance between pro- and anti-inflammatory factors release.