Heat shock protein responses to aging and proteotoxicity in the olfactory bulb

Abstract

The olfactory bulb is one of the most vulnerable brain regions in age-related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here, we describe age-related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2-4 months. To model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro-oxidant paraquat. Toxin-induced increases were observed in Hsp70, Hsp25, and Hsp32. To determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells. The olfactory bulb is affected in the early phases of many age-related neurodegenerative disorders. Here, we described the impact of aging on multiple heat shock proteins (Hsps), such as Hsp70, in the female rat olfactory bulb in vivo. Using multiple proteasome and Hsp70 inhibitors (see schematic), we found that proteotoxicity elicited a compensatory increase in Hsp70 in primary olfactory bulb cells in vitro. Hsp70 then reduced the proteotoxic buildup of ubiquitinated proteins and robustly protected against cell death according to three independent viability assays. Thus, olfactory bulb neurons can mount impressive natural adaptations to proteotoxic injury, perhaps explaining why neurodegenerative disorders are so delayed in onset and so slow to progress.

Keywords: Glucose-regulated protein 78; heat shock cognate 70; heat shock protein 70; heme oxygenase 1; olfaction; proteostasis.

Figure 1. Impact of aging on Hsps and co-chaperones in the olfactory bulb of the female rat

(A-E) Infrared Western immunoblots are shown for the indicated Hsps and co-chaperones. Olfactory bulb tissue was harvested from female rats at 2.0-3.9 months, 4.0-6.0 months, 8.0-9.0 months, 16.0-18.9 months, and 19.0-22.0 months of age. GAPDH, α-tubulin, or β-actin was used as a protein loading control, depending on the species of the primary antibodies and the expected molecular weights. n = 3-6 rats per group. * p ≤ 0.05, ** p ≤ 0.01, versus 2-4 month old, + p ≤ 0.05, ++ p ≤ 0.01 versus 4-6 month old, ~ p ≤ 0.05, ~~ p ≤ 0.01 versus 8-9 month old, LSD post hoc following one-way ANOVA.

Figure 2. Primary postnatal cultures of the olfactory bulb

Dissociated primary olfactory bulb cells from day 1-2 neonatal rat pups were stained on day-in-vitro 5 (DIV5) for the neuronal marker MAP2, the synaptic protein synaptophysin, the astrocyte marker GFAP, and the Hoechst nuclear stain. Control wells (bottom panels) were exposed to all solutions except the primary antibodies.

Figure 3. In vitro model of proteotoxic and oxidative stress in olfactory bulb cultures

Primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin (A-E) and MG132 (F-J), and the redox-active herbicide paraquat (K-O) on DIV5 and assayed by three independent assays for cell viability on DIV7: the infrared DRAQ5 stain for nuclei (A, D, F, I, K, N), infrared In-Cell Western analyses for the neuronal marker MAP2 (B, E, G, J, L, O), and the Cell Titer Glo assay for ATP (C, H, M). Representative DRAQ5 and MAP2 images were pseudocolored red and green, respectively. Shown are the mean and S.E.M of 3 independent experiments, each performed in triplicate. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 versus vehicle-treated groups, Bonferroni post hoc following one-way ANOVA.

Figure 4. Mild proteotoxic and oxidative stress increases Hsps in olfactory bulb cultures

(A-E) Primary olfactory bulb cultures were treated with low concentrations of lactacystin, MG132, and paraquat on DIV5 and harvested for measurements of Hsps on DIV6 by infrared Western blotting. GAPDH or β-actin was used as a protein loading control. Shown are the mean and S.E.M of 3-6 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 versus vehicle-treated groups, two-tailed t-test.

Figure 5. Effect of Hsp70/Hsc70 and HO1 inhibition on proteotoxic stress in olfactory bulb cultures

Primary olfactory bulb cultures were treated on DIV5 with low concentrations of lactacystin (A-C) or MG132 (D-F) in conjunction with the Hsp70/Hsc70 inhibitor VER155008 or the HO1 inhibitor tin protoporphyrin (SnPPx) and three independent viability assays were performed on DIV7. Representative infrared images are shown in G and H. (I-M) Primary olfactory bulb cultures were treated on DIV5 with low concentrations of MG132 in conjunction with the Hsp70/Hsc70 inhibitor MAL3-101 and three independent viability assays were performed on DIV7. Shown are the mean and S.E.M of 3-6 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 versus 0 μM MG132 or lactacystin; + p ≤ 0.05, ++ p ≤ 0.01, +++ p ≤ 0.001 versus 0 μM VER155008, MAL3-101, or SnPPx, Bonferroni post hoc following two-way ANOVA.

Figure 6. Impact of mild stress and Hsp70 activity on ubiquitinated protein levels in olfactory bulb cells

(A) Olfactory bulb tissue was harvested from female rats at 2.0-3.9 months, 4.0-6.0 months, 8.0-9.0 months, 16.0-18.9 months, and 19.0-22.0 months of age and probed for ubiquitin-conjugated proteins by infrared Western blotting. n = 3-6 rats per group. (B) Primary olfactory bulb cultures were treated with low concentrations of lactacystin, MG132, and paraquat on DIV5 and harvested for immunoblots of ubiquitinated proteins on DIV6. Shown are the mean and S.E.M from 5-6 independent experiments. (C-D) Primary olfactory bulb cultures were treated on DIV5 with MG132 in the absence or presence of the Hsp70/Hsc70 inhibitor VER155008 and assayed for pan-ubiquitinated proteins (C) or K48-linked ubiquitin (D) on DIV6. GAPDH or β-actin was used as the loading control. Shown are the mean and S.E.M from 5-6 independent experiments. *** p ≤ 0.001 versus 0 μM MG132; +++ p ≤ 0.001 versus 0 μM VER155008, Bonferroni post hoc following two-way ANOVA.