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. In order 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. In order 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 dual hit hypothesis of neurodegeneration states that severe stress sensitizes vulnerable cells to subsequent challenges so that the two hits are synergistic in their toxic effects. Although the hippocampus is vulnerable to a number of neurodegenerative disorders, there are no models of synergistic cell death in hippocampal neurons in response to combined proteotoxic and oxidative stressors, the two major characteristics of these diseases. Therefore, we developed a relatively high-throughput dual hit model of stress synergy in primary hippocampal neurons. In order to increase the rigor of our study and strengthen our interpretations, we employed three independent, unbiased viability assays at multiple timepoints. Stress synergy was elicited when hippocampal neurons were treated with the proteasome inhibitor MG132 followed by exposure to the oxidative toxicant paraquat, but only after 48h. MG132 and paraquat only elicited additive effects 24h after the final hit and even loss of heat shock protein 70 activity and glutathione did not promote stress synergy at this early timepoint. Dual hits of MG132 elicited modest glutathione loss and slightly synergistic toxic effects 48h after the second hit, but only at some concentrations and only according to two viability assays (metabolic fitness and cytoskeletal integrity). The thiol N-acetyl cysteine protected hippocampal neurons against dual MG132/MG132 hits but not dual MG132/paraquat hits. Our findings support the view that proteotoxic and oxidative stress propel and propagate each other in hippocampal neurons, leading to synergistically toxic effects, but not as the default response and only after a delay. The neuronal stress synergy observed here lies in contrast to astrocytic responses to dual hits, because astrocytes that survive severe proteotoxic stress resist additional cell loss following second hits. In conclusion, we present a new model of hippocampal vulnerability in which to test therapies, because neuroprotective treatments that are effective against severe, synergistic stress are more likely to succeed in the clinic.